Method for diagnosing, preventing, and treating neurological diseases

ABSTRACT

The invention provides for methods of treating autism associated with  Desulfovibrio  overgrowth in the gastrointestinal tract of a patient, said method comprising administering to the patient suffering from said autism a treatment course of aztreonam in an amount effective to treat autism in the patient, thereby treating autism.

This patent application claims the benefit of the filing date of U.S.Ser. No. 61/275,714, filed May 26, 2010, the contents of all of whichare herein incorporated by reference in their entireties into thepresent patent application.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to treatment of neurological diseases,especially autism. The treatment involves antibiotics and repopulatingthe gastrointestinal tract with normal flora and also vaccines andbacteriophage.

BACKGROUND OF THE INVENTION

The composition of the normal gastrointestinal flora varies somewhatfrom individual to individual. Some bacterial species may be carriedonly transiently, but most are fairly permanent. Some members of thenormal flora can become pathogenic if they acquire additional virulencefactors (e.g., E. coli) or are introduced into normally sterile sites(e.g., Staphylococcus aureus). Normal flora is generally beneficial—forexample, the normal flora may prevent pathogenic microorganisms fromproliferating in the body (a phenomenon known as colonizationresistance), and may also produce essential nutrients (e.g., vitamin Kis produced by the gut flora).

The use of antibiotics is ubiquitous among children and adults forbacterial infections, and they are often also prescribed for viralinfections. This prolific use has come under criticism for variousreasons, most notably for inducing microbial resistance to previouslyeffective antibiotics and rendering them less effective or ineffectiveagainst dangerous human pathogens. For example, multidrug-resistantstrains of Mycobacterium tuberculosis seriously threaten tuberculosis(TB) control and prevention efforts. Administration of broad-spectrumantibiotics has a profound effect on the normal flora and can result incolonization with antibiotic-resistant organisms. Antibiotic-mediateddisruption of the normal flora can lead to fungal infections, such asinvasive candidiasis, or to antibiotic-associated colitis caused byClostridium difficile.

Several neurological or neuropsychiatric conditions, such as autism mayhave gastrointestinal etiology. Published data lend credence to thenotion that an alteration in bowel microflora may be associated withautistic symptoms (Sandler et al., J. Child Neural. 15, 429-435, 2000;Finegold et al., Clin. Infect. Dis. 35 (Suppl.1), S6-S16, 2002; Song etal., Appl. Environ. Microbial. 70, 6459-6465, 2004; Parracho et al., J.Med. Microbial. 54, 987-991, 2005; Finegold et al., Medical Hypotheses70, 508-511).

SUMMARY OF THE INVENTION

The invention relates to methods for preventing or treating agastrointestinal or neurological disorder. The disorders preferably haveas an etiological component a microbial agent. The method comprisesadministering to the patient an antimicrobial composition in an amounteffective to inhibit or eliminate the microbial agent. By “microbialagent” is meant a microbe or its toxin. Disorders that can be treated bythe methods of the invention include attention deficit disorder,depression, bipolar disorder, juvenile diabetes, primary sclerosingcholangitis, Alzheimer's disease, Parkinson's disease, Whipple'sdisease, Tourette's syndrome, juvenile rheumatoid arthritis, adultrheumatoid arthritis, multiple sclerosis, Asperger's syndrome, pervasivedevelopment disorder, autism (especially early onset and regressiveautism), Rhett's syndrome, D-lactic acidosis, obesity, atherosclerosisand atherosclerotic heart disease, chronic fatigue syndrome, Gulf Warillness, post-traumatic stress disorder and schizophrenia.Gastrointestinal disorders can include antimicrobial associated diarrheaor inflammatory bowel diseases such as ulcerative colitis or Crohn'sdisease. The method can be used where the agent is a species of thegenus Clostridium or Desulfovibrio or other genera that are abnormal inthe intestinal flora. Colon cancer may also be related to the presenceof such organisms in the bowel and so may be prevented by eliminatingsuch organisms. As used herein, “abnormal” refers to organisms that 1)are not normally present in the intestinal flora; 2) are present insignificantly higher or lower concentrations than in the normal flora;3) produce one or more toxic products not produced by organisms of thenormal flora; 4) compete with the host for essential nutrients or otherelements; and/or 5) produce disease in people with abnormal immunesystem.

The antimicrobial composition preferably has at least one of thefollowing properties: oral palatability, sustained concentration in thegastrointestinal tract, low absorption from the gut (and hence lowsystemic concentration), higher activity against the abnormal organismrelative to activity against other normal gut flora, bactericidalactivity, not cross-resistant with vancomycin or other antimicrobialsthat are important for treatment of systemic infections, resistance doesnot develop readily, the composition is well tolerated orally and overan extended period of time (preferably at least 3-4 months), it iseffective when given once or twice daily, has low systemic andgastrointestinal toxicity, and is economical. Available in liquid formfor subjects unable to swallow pills or tablets.

An alternative or supplemental therapy involves the use of abacteriophage in addition to or as the antimicrobial composition. Thebacteriophage is preferably specific for the abnormal organism.

Another alternative or supplemental method of treating a neurological orgastrointestinal disorder is a therapy regimen to repopulate thegastrointestinal tract with normal flora. This therapy comprisesadministering to the patient at least one of the normal gut inhabitantsthat is present in healthy people in high numbers by any route. Thismethod can be used in conjunction with the antimicrobial composition.Preferably, the antimicrobial is first administered to suppress oreradicate the abnormal organism, then the normal flora is repopulated bythe administration of at least one of the normal gut inhabitants. It ispreferred that the antimicrobial treatment is complete before theadministration of the at least one of the normal gut inhabitants.

In another embodiment, the invention includes a method of detecting aneurological or gastrointestinal disorder that has as an etiologicalcomponent an abnormal microorganism in the intestinal flora. The methodcomprises collecting a gastrointestinal sample from a patient suspectedof having such disorder, and screening for the presence or concentrationof the abnormal organism and/or certain toxic substances. This methodcan be used with other techniques to diagnose the presence of autism ina patient, such as observation of lack of eye contact, difficulty withsocial relationships, speech delays, or odd physical behaviors.Screening instruments have been developed to quickly gather informationabout a child's social and communicative development within medicalsettings. Current diagnosis relies on checklists of symptom whichinclude the Checklist of Autism in Toddlers (CHAT), modified Checklistfor Autism in Toddlers (M-CHAT), the Screening Tool for Autism inTwo-Year-Olds (STAT), and the Social Communication Questionnaire (SCQ,for children 4 years of age and older).

Alternatively, if the abnormal organism produces a toxin, the sample canbe screened with an antibody directed against a conserved epitope of thetoxin, where a specific interaction of the antibody with the sampleindicates the presence of a neurological disorder in the patient. Analternative embodiment is the use of an antibody generated against thespecific toxin causing the neurological or gastrointestinal disorder.Such an antibody can be produced by conventional means (e.g.,polyclonal, monoclonal), or can be derived from a patient having a highserum titer to the causative agent.

Another feature of the invention is a method of treating or preventing aneurological or gastrointestinal disorder in a patient, the disorderhaving as an etiological component a microbe that produces a toxin, themethod comprising vaccinating the patient with an antigenic epitope ofthe toxin such that an immune response capable of interaction with gutflora (e.g., via Peyer's patches, IgA, or other, immunoglobulin orcomplement activation local to the gut) can be elicited upon antigenchallenge from microbe proliferation in the gut.

The invention also provides for methods for treating autism associatedwith Desulfovibrio overgrowth in the gastrointestinal tract of apatient, said method comprising administering to the patient sufferingfrom said autism a treatment course of aztreonam in an amount effectiveto treat autism in the patient, thereby treating autism.

The invention further provides for methods for screening for compoundsthat inhibit Desulfovibrio and thereby inhibit autism, wherein saidmethod comprises obtaining a sample from a subject, contacting a samplecontaining Desulfovibrio with a molecule of interest, and determiningwhether contact results in inhibition of Desulfovibrio.

The invention further provides for methods of screening for autismcomprising obtaining a sample from a subject, and determining whetherDesulfovibrio is present in the sample, the presence of Desulfovibriobeing indicative of autism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pie chart showing the frequencies of bacterial phyla in thestool of autistic children (FIG. 1A), control children (FIG. 1B) andsiblings of autistic children (FIG. 1C).

FIG. 2 is a graph of the Principal Component Analysis at the phylumlevel. FIG. 2A shows the mapping according to the severity of theautism. FIG. 2B shows phylum level mapping of the autistic children (A),control (C), and sibling (SC).

FIG. 3 is graph of the Principal Component Analysis at the genus level.FIG. 3A shows the mapping according to severity of autism. FIG. 3B showsgenus level mapping of the autistic children (A), control (C), andsibling (SC).

FIG. 4 is a map of the genus abundance analysis.

FIG. 5 shows a diagram of the proposed pathogenesis of autism.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for treating autism associated withDesulfovibrio overgrowth in the gastrointestinal tract of a patient. Themethod comprises administering a treatment course of aztreonam in anamount effective to treat autism in the patient. In a furtherembodiment, the method comprises administering a beta-lactamaseinhibitor selected from clavulanic acid, tazobactam, sulbactam andLK-157 or others.

In one embodiment, the aztreonam and the beta-lactamase inhibitor areadministered concurrently.

In another embodiment, the invention further comprise administering aprobiotic and/or a probiotic group. Examples of probiotic includes butnot limited to bacteria (a single or multiple species) that competeswith Desulfovibrio for nutrients and bacteria that inhibits growth ofDesulfovibrio. A more detailed description is found in the section“Probiotic Therapy”.

In another embodiment, the subject (or patient) is selected from human,monkey, ape, dog, cat, cow, horse, rabbit, mouse and rat subjects. In afurther embodiment, the subject is human.

It has been discovered that disruption of gastrointestinal flora orpoorly developed gastrointestinal flora in young infants and subsequentpathogenic microbial proliferation in one or more regions of thegastrointestinal tract can mediate a variety of disruptions ofneurological function. These neurological disruptions are mediated bytoxins, or profound metabolic disturbances related to the metabolism ofthe offending organism (e.g. sulfate metabolism problems related to theutilization of sulfate by Desulfovibrio) which extends to disrupting theprotective mucin layer of the GI tract which is made up of sulfatedglycoprotein which can result in inflammation and increased permeabilityof the gut particularly neurotoxins, produced by one or more species ofthe proliferating microbes. Bacteria of several genera are indicated asthe likely causative agents and/or their toxins. The toxins can bepotent, non-necrotizing neurotoxins that disrupt neurotransmitterrelease. Neurotoxins such as the neuromuscular transmission inhibitortetanospasmin, produced by C. tetani, have traditionally been consideredto be dangerous following systemic exposure only (e.g., exposure byintroduction of the bacterium or spores into a wound, for example). Ithas also been discovered that patients of certain neurological conditionhave an intestinal flora that is different from the normal flora. As aspecific example, when compared to the normal flora, the intestinalflora of autistic patients contains a significantly greater percentages(based on the total flora) of Desulfovibrio. As such, the presence of anabnormal Desulfovibrio concentration can be indicative of autism.

It has also been discovered that the methods of the invention, includingantibiotic therapy directed to the abnormal microorganism, results inimproved neurological function through inhibition or elimination of theproliferating species. Recurrence of the neurological symptoms can belimited or prevented by repopulation of the gastrointestinal tract bynormal human gut flora (“probiotic therapy” or fecal transplantation) orby administration of certain foodstuffs to stimulate growth of thedesirable bacteria (“prebiotic therapy”). The neurological symptomsthemselves can be prevented or limited in the place by appropriateprobiotic or prebiotic therapy following administration of wide spectrumantibiotics, especially in children or compromised adults.Alternatively, limiting the use of broad-spectrum antibiotics can alsohelp prevent the widespread disruption of the gastrointestinal flora atthe outset. Finally, vaccination leading to a gut level immunoresponseagainst toxin antigens responsible for the neurologic symptoms can beused to prevent occurrence of disorders due to microbial overgrowth.

The pathogenic proliferation of microbes in the gut can at leastpartially cause deleterious neurological symptoms and syndromes of manydisorders, including at least some forms of pervasive developmentdisorder including autism (both early-onset and late-onset autism);Asperger's syndrome; attention deficit disorder; depression; bipolardisorder; Alzheimer's disease; Parkinson's disease; Whipple's disease;Tourette's syndrome; Rhett's syndrome; and schizophrenia.

Additional diseases and disorders are also caused by disrupted gutmicrobial flora, and can be treated and/or prevented by antibiotic andprobiotic or prebiotic measures. Examples of such disorders includeantimicrobial-associated diarrhea and inflammatory bowel disease(including, for example, ulcerative colitis and Crohn's disease). A veryimportant example is hospital-acquired (nosocomial) systemic infectiondue to S. aureus, Pseudomonas, Klebsiella-Enterobacteria, etc. which mayoften be traced to previous colonization of the intestinal tract bythese organisms following hospitalization.

Many, if not most, of the disorders mentioned above are clearlyunrelated by conventional medical knowledge and/or standards. However,the surprising discovery of a common etiology makes possible a commondiagnostic, therapeutic, and preventative concept, embodied in themethods of the invention.

Abnormal Microorganism

The present invention targets members of several genera as causative orcontributing to the neurological or intestinal disease. The genera,including Clostridium, Desulfovibrio, Bacteroides, Turicibacter,Weissella, Parabacteroides, and Ruminoccocus. Clostridium are plausibleas agents of these neurological disorders, because several members ofthis genus are known to produce neurotoxins, they proliferateenterically during antimicrobial therapy (e.g., C. difficile), and theyhave been implicated in diarrheal diseases of humans and animals. Forexample, Clostridium tetani is the bacterium that causes tetanus(lockjaw) in humans. C. tetani spores can be acquired from soil or anytype of skin trauma involving an infected device. If an anaerobicenvironment is present, the spores will germinate and eventually formactive C. tetani cells. At the tissue level, the bacterium then releasesan exotoxin called tetanospasmin that causes certain nervous systemirregularities by means of retrograde transmission through neurons tothe nervous system. One of the toxin's classic effects includes constantskeletal muscle contraction due to a blockage of inhibitory interneuronsthat regulate muscle contraction. Prolonged systemic infectioneventually leads to respiratory failure, among other things. If nottreated early, the mortality rate of this disease is high. Botulism isanother disease related to clostridia (C. botulinum) and infant botulismis caused by GI colonization with this organism acquired from food orsoil and can be precipitated or exacerbated by antibiotic therapy.

Desulfovibrio is a gram negative, rod shaped, sulfate reducingbacterium, which is an anaerobe and which may compete with the host forsulfur. Its metal corroding ability has led to numerous health andsafety concerns; and its production of H₂S and endotoxin can lead togenotoxic and other toxic-related problems.

Although several genera, as exemplified above, are generally culprits inthe gastrointestinal component of neurological disorders, other abnormalorganisms may also be involved. The other abnormal organisms, however,and their association with specific diseases/conditions can beelucidated using the methods described in this document.

Recognition of the gastrointestinal component of these neurologicaldisorders allows diagnosis and treatment of a novel and specific nature.

Diagnosis

The invention also provides methods for screening for compounds thatinhibit Desulfovibrio and thereby inhibit autism. Method for screeningfor compounds comprises obtaining a sample from a subject, contacting asample containing Desulfovibrio with a molecule of interest, anddetermining whether contact results in inhibition of Desulfovibrio.

In one embodiment, the inhibition of Desulfovibrio includes decreasinggrowth of Desulfovibrio, eliminating growth of Desulfovibrio or delayinggrowth of Desulfovibrio.

In one embodiment, the compound is bacteriostatic or bactericidal. Inanother embodiment, the compound is a small molecule, protein, peptide,antibiotic, pre-formed antibodies, bacteriophage or a combinationthereof.

In one embodiment, the screening method comprises separately contactingeach of a plurality of samples to be tested. In another embodiment, theplurality of samples comprises more than about 10⁴ samples. In a furtherembodiment, the plurality of samples comprises more than about 5×10⁴samples.

In another embodiment, the sample is selected from feces, urine, bloodand plasma.

The invention further provides a method of screening for autismcomprising obtaining a sample from a subject, and determining whetherDesulfovibrio is present in the sample, the presence of Desulfovibriobeing indicative of autism.

Diagnostic options include tests based on detection of the organismitself (culture or PCR), the gene that codes for the toxin produced bythe overgrown microbe, the toxin itself, or proliferation of aparticular toxin-producing microbe. Such tests include amplification ofnucleic acids encoding the toxin (e.g., by PCR, using primers/probesspecific for the toxin or toxins of interest; specific hybridizationassays (e.g., in situ hybridization; Northern, Southern, or dot blots;microarray hybridization, etc.); detection of the toxin itself using,e.g., anti-toxin antibodies, (generated monoclonally, polyclonally, orderived from patients with high titers of anti-toxin antibodies (suchantibodies are likely to be better reagents than commercial tetanusantibodies or antibodies generated against conserved regions of multiplebacteria, since they will be exceptionally specific for the causativetoxin) in ELISAs, sandwich assays, Western blots, or affinitychromatography; animal assays; laser mass spectroscopy, or any othermethods known to those of skill in the art. Samples can be obtained fromfecal samples, blood, plasma, urine, saliva, cerebrospinal fluid, biopsytissue, or any other patient source, and may be directly tested or afterisolation of suspected causative agents.

Screening assays are based on detection of suspect organisms in thefeces of patients using culture (sometimes employing enrichment,selective and/or differential media) and microbiologic and molecularidentification techniques, including immunofluorescent techniques,genetic probes, laser mass spectroscopy, or other methods known in theart.

The methods of diagnosis herein can be used in conjunction with or tosupplement other diagnostic methods known in the art. For example,diagnosis for autism may include interviews with parents/caretaker ofthe patient or observation of the patient.

Selection of Antimicrobial Therapeutic Agents

Antimicrobials to Treat Disorders Resulting from Disrupted Gut Flora

Once a positive diagnosis has been made, antimicrobial therapy can bestarted to inhibit or eliminate the microbe whose enteric overgrowthand/or toxin production is causing the disorder. The antimicrobials usedto treat the disorders described above should have certaincharacteristics for optimal benefit and minimal side effects. Certainantimicrobials have characteristics appropriate to treat even very youngchildren, and such drugs are useful to treat disorders having thegut-brain involvement. Preferably, an antimicrobial selected as atherapy for any of the above disorders will have one or more of thefollowing properties:

-   -   1. Good in vitro activity against most or all clostridial        species and/or Desulfovibrio;    -   2. Relatively poor activity against most other organisms        normally found in the gut flora;    -   3. Safe doses capable of achieving a concentration in the colon        or elsewhere in the GI tract where the offending organism        proliferates exceeding the minimal inhibitory concentration or        minimal bactericidal concentration of the drug by at least four        or five-fold concentrations;    -   4. Preferably absorbed very little or not at all when given        orally (to minimize systemic effects);    -   5. Bactericidal activity preferred (rather than purely        inhibitory activity);    -   6. Not cross-resistant with vancomycin or other drugs that are        important for treatment of systemic infections;    -   7. Resistance doesn't develop readily: (i.e., the drug doesn't        readily engender resistance in bacteria);    -   8. Palatable in liquid form when taken orally (for        administration to children), or readily formulated into other        oral doses (to enhance patient compliance);    -   9. Well tolerated orally over extended period of time        (preferably at least 3-4 months);    -   10. Little or no toxicity, either systemically or in the bowel;    -   11. Preferably effective when given only once or twice daily;        and    -   12. Preferably moderate in price.

Drugs that have one or more of the above characteristics may haveutility for antimicrobial therapy in treating neurological disorderswith a gut flora etiology include those listed below:

ABT-773 Aminoglycosides Ampicillin/sulbactam Amphomycin AzithromycinAztreonam Bacitracin Beta-lactamase inhibitors CarbomycinCephalosporins, oral Clarithromycin Colistin Erythromycins Fidaxomycin(OPT 80) Furazolidone, other nitrofurans Fusidic acid, Na fusidateGramicidin Imipenem, oral; other penems Josamycin Linezolid, otheroxazolidinones Macrolides Metronidazole, other nitroimidazoles MikamycinNovobiocin Oleandomycin, triacetyloleandomycin OstreogrycinPiperacillin/tazobactam Polymyxin Pristinamycin Ramoplanin RifaximinRistocetin Rosamicin, rosaramicin Spectinomycin Spiramycin StaphylomycinStreptogramin Synergistin Teicoplanin Telavancin TelithromycinTicarcillin/clavulanic acid Tyrocidin Tyrothricin Vancomycin VernamycinVirginiamycin

Appropriate doses of these antimicrobials are within the range given formany other conditions for which the antimicrobials are prescribed.Dosage information can be found, for example, in the Physicians' DeskReference, 54th Edition, Medical Economics Company, Montvale, N.J.(2000). In certain instances, the doses may be elevated to the extentnecessary to maintain a bactericidal or bacteriostatic concentrationthroughout the gastrointestinal tract. The antimicrobials are preferablyformulated for oral administration, such as in liquid form, tablet,capsule, granules, chewable, etc. Tablets or capsules may be entericallycoated to minimize gastric absorption of the drug (since very fewbacteria are capable of colonizing the stomach, this is not necessarilya primary target of the therapies of the invention). However, when thepH of the stomach is high or emptying is slow due to certain drugs ordiseases, the stomach can be colonized with many bacteria.

The antimicrobials can be administered as known in the art. It isdesirable to select a route of administration that is most effective forthe therapy, examples thereof being oral administration or parenteraladministration such as intravenous administration.

A preferred compound for treating Clostridium overgrowth in the gut isramoplanin, also known as A-16686 (see, e.g., U.S. Pat. Nos. 4,303,646;4,328,316; 4,427,656; 5,539,087; and 5,925,550; and Parenti et al.;Drugs Exp Clin Res 16(9):451-5 (1990); all herein incorporated byreference). This antibiotic is not cross-resistant with vancomycin, itengenders very little to no resistance in bacteria, is not detectablyabsorbed systemically in humans (making it exceptionally safe, even foryoung children), can be made palatable in a liquid form, achieves highconcentrations in the large intestine, has very good activity againstclostridia, can be given twice a day, and is primarily active againstgram positive organisms at the dosage levels administered. Ramoplanin ispreferable to drugs such as vancomycin and metronidazole, which havepreviously been used, because, for example, vancomycin, while achievinga high concentration in the intestines throughout, is effective againstBacteroides, a beneficial genus of gut flora, as well as clostridialspecies. It is also a potent antibiotic against, e.g., systemicmethicillin-resistant Staphylococcus infections, and widespread use forother purposes risks inducing vancomycin-resistant Staphylococcusspecies. Metronidazole, on the other hand, is not an ideal candidatebecause of its ready systemic absorption, which can lead to neurotoxicside effects when given in high enough concentrations to remaineffective in the gut, and the fact that it is quite bitter and thusdifficult to formulate as a liquid for oral use.

Therapies to Prevent Occurrence of Pathogenic Bacterial Overgrowth andAttendant Disorders

It is desirable to prevent, rather than merely treat, thegastrointestinally mediated neurological disorders discussed herein, byreducing the extent of normal bacterial disruption in the gut duringantimicrobial treatment for other infections. This can be done by notusing antibiotics for viral or other non-bacterial infections, but if anantibiotic must be used, it should be tailored as specifically aspossible against the identified or most likely causative agent.

For example, one common drug to avoid in treating infections in youngchildren is trimetoprim/sulfamethoxazole because it has been anecdotallyindicated by parents of late onset autistic children as a commonbackground factor (use of this antimicrobial for, e.g., ear infections,just prior to onset of autistic symptoms). This drug has also been shownto cause major overgrowth of clostridia in the bowel flora of adults(see, e.g., Haralambie et al., Infection 11(4):201-4 (1983). On theother hand, in the methods of the invention, a drug such as ampicillinmay have a good spectrum of activity against the pathogens of otitismedia (principally Streptococcus pneumoniae and Haemophilus influenzae)and is also active against clostridia, so would not likely to lead toovergrowth of clostridia in the bowel flora. It may need to be combinedwith a β-lactamase inhibitor such as sulbactam.

Another embodiment of the invention that may be considered is thealternative medicine approach in which phytonutrients (plant products)such as curcumin or hops may be used with good effect and little or notoxicity.

It is important to use agents with as narrow and specific a spectrum aspossible for the disorder being treated. A different or supplementalapproach (discussed more fully below) is to replenish the eliminatedflora as quickly as possible with probiotic or prebiotic treatment toprevent overgrowth of the problem clostridia.

Another embodiment of the invention is to immunize children in such away that they obtain immunity at the level of the gut mucosa to thetoxin involved. This involves eliciting an immunoglobulin responsespecific against exposed antigens of the Clostridium or Desulfovibriotoxin or toxins. Cell-mediated immunity is also important in mucosalimmunity to various pathogens (van Ginkel et al., Emerging Infect. Dis.,6:123-132, 2000. The pathogenic effect of overgrowth of the bacterialspecies involved (those producing the neurotoxins), even if it occurs,is then rendered harmless by the immune response against the toxinlocally, at the gut where the toxin is produced. Eliciting this response(e.g., via B cells aggregated in the Peyer's patches/lymph nodes of theintestine) involves an antitoxin to the toxin, toxoid, or modified toxinthat would induce immunity to the toxin. The data provided in theExamples below demonstrate that one or more toxins with homology totetanus toxin (tetanospasmin) are responsible for the neurologicalsymptoms seen in, e.g., late onset autistic children, and a region ofhigh homology among two or more toxin genes is the preferable region orepitope to use to induce the antigenic response.

Since tetanus toxin is a member of the family of zinc endopeptidases,the use of a selective synthetic or natural zinc endopeptidase inhibitoris also a therapeutic option to reverse or prevent the neurologicaleffects of chronic or subacute Clostridium infection and resultant toxinrelease. Examples of pseudotripeptide compounds useful in this respect,containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety asthe P1 side chain and natural amino acids in the P1′ and P2′ components,can be found in Martin et al., J. Med. Chem., 42(3):515-525 (1999),herein incorporated by reference. Captopril, an oral medication welltolerated by children, is such an inhibitor and inactivates tetanustoxin in vitro.

As a last resort, surgical or pharmacologic vagotomy may be used inespecially refractory cases of neurologic disorder caused by clostridialneurotoxin. The rationale is that tetanus toxin is known to travelretrogradely up the vagus nerve (which innervates the gastrointestinaltract), and vagotomy would prevent transmission of toxin from the gut tothe brain, thus alleviating the neurological symptoms and preventingrecurrence.

Probiotic Therapy

A preferred therapy, however, alone or in conjunction with one or moreof the therapies discussed herein, is probiotic therapy. “Probiotic”therapy is intended to mean the administration of organisms andsubstances which help to improve the environment of the intestinal tractby inhibiting the disproportional growth of bacteria which producetoxins in the intestinal tract. For example, in healthy humans, thebowel is colonized by Bifidobacterium, lactobacilli (e.g., L.acidophilus), gram-negative anaerobes, enterococci, Bacteroides sp.,Parabacteroides, Prevotella, Porphyromonas, gram-positive anaerobiccocci, Clostridium sp., Enterobacteriaceae (mainly E. coli), andenterococci and other less well-known bacteria. Some of these bacteriaproduce substances which suppress harmful bacteria; for example,bifidobacteria produce lactic and acetic acid, decreasing the pH of theintestines. They can also activate macrophages, which also help suppressharmful bacteria.

The best strains for supplementation are those that are typicallypermanent residents of the human intestinal tract and which do notproduce toxins. Normal human intestinal flora are better adapted to theenvironment (bile acids, anaerobic conditions, etc.) of the humanintestinal tract, and are more likely to survive and colonize the humanintestinal tract. Certain species such as L. bulgaricus and S.thermophilus, for example, are commonly used as probiotics, but are notnormal constituents of human gut flora, and such species apparently donot colonize the intestinal tract well.

The probiotic therapy of the invention is designed to be administered asa mixture of a number of species that are normal, benign inhabitants ofthe gut, preferably in the general proportion in which they are found inhealthy humans. For example, E. coli is a common enteric inhabitant, butmakes up only about 1/1000 of the bowel flora found in healthy humans,so would be a relatively small proportion of a probiotic mixture.Description of normal human gut flora and relative abundances can befound in Tables 1-2 below, Finegold (J. Assoc. Anaerobic. Infect. Res.28:206-213 (1998), and Finegold et al. (Normal Indigenous IntestinalFlora, Chap. 1, in Hentges, D. J., ed. Human Intestinal Microflora inHealth and Disease, New York, Academic Press, p. 3-31, 1983; which areincorporated herein by reference.

TABLE 1 Prevalence of major organisms in fecal flora Mean % StoolsCount/gm Positive (Log₁₀) Gram-negative anaerobic rods 100 11.3Gram-positive NSF* anaerobic rods 99 11.1 Anaerobic cocci 94 10.7Clostridium 100 9.8 Streptococcus 99 8.9 Gram-negative aerobic orfacultative rods 98 8.7 Other aerobic or facultative organisms 93 6.8*NSF = Nonsporeforming

TABLE 2 Most prevalent species in fecal flora Mean % Stools Count/gmPositive (Log₁₀) Bacteroides thetaiotaomicron 87 10.7 Bacteroidesvulgatus 70 10.6 Bacteroides distasonis 53 10.5 Bacteroides fragilis 4610.4 Bifidobacterium adolescentis group 55 10.0 Eubacterium aerofaciens49 9.7 Clostridium ramosum 53 9.1 Escherichia coli 93 8.6 Streptococcusfaecalis group 80 7.5

A suitable probiotic mixture is composed of at least one, preferably atleast three, more preferably a larger number, of the species listed inTable 2 and others in about the proportions found normally in the colon(see list in the “Mean Count/gm” column). It is estimated that, in all,there may be 300-400 species found in human colonic flora and recentresearch suggests 1,000 or more species.

Dosage (colony forming units (cfu) of each bacterium) is preferably atleast the number found in the mean count/gram, and is supplied to thepatient daily or twice daily for a number of days until it is determinedthat the bacteria have become established. The formulation can beprovided as active cells or spores. It can be provided in an entericcoated form (e.g., for active cells) to protect sensitive cells from thegastric environment. A preferred therapy involves temporary eliminationor suppression of the patient's flora (primarily or entirely with theuse of antimicrobial agents) and introduction of a new, non-pathogenicflora that consists of a number of bacteria normally found in the bowelthat convey colonization resistance (to prevent regrowth orre-implantation of the offending bacteria). Therapies are preferablypatterned after those described in the poultry literature, for example,Wooley et al., Avian Dis. 43(2):245-50, (1999); Hume et al., J. FoodProt. 61(6):673-6 (1998); Corrier et al., J. Food Prot. 61(7):796-801(1998); Hume et al., Avian Dis. 40(2):391-7 (1996); Cornier et al.,Poult Sci. 74(7):1093-101 (1995); and Corrier et al., Poult Sci.74(6):916-24 (1995), all herein incorporated by reference.

Alternatively, bacteriophage specific for the bacterium producing thetoxin can be introduced to the patient's gastrointestinal tract toreduce or kill the toxin-producing bacteria, and probiotic therapymixtures can be concurrently or subsequently administered. An example ofa successful protocol involving this strategy with Clostridium difficilecan be found in Ramesh et al., Anaerobe 5:69-78 (1999), hereinincorporated by reference. Bacteriophage may be susceptible to gastricacidity and such acidity should be neutralized prior to phageadministration, or else the bacteriophage can be administered in anenterically coated tablet or capsule.

Probiotic and/or prebiotic therapy can be used in conjunction withantimicrobials used to treat infections in otherwise normal patients(i.e., before the onset of a neurological disorder) in order to preventor reduce the risk of the occurrence of a neurological disorder.Alternatively, it can be used in conjunction with antimicrobials beingused to eliminate or inhibit the abnormal microorganism(s) in apatient's gastrointestinal tract, and to promote the re-emergence ofnormal gut flora and proportions/balance. It is preferred that theprobiotic is administered orally.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and use the present invention. The followingexamples are given to illustrate the present invention. It should beunderstood that the invention is not to be limited to the specificconditions or details described in these examples.

EXAMPLES Example 1 Results in Autistic Children

Experiments conducted with late-onset autistic children havedemonstrated success using methods of the invention. The inventors haverecorded significant improvement in the symptoms of children withdelayed-onset autism by providing them with antibiotics directed towardcommon anaerobic intestinal bacteria. By “delayed-onset,” “regressive,”or “late onset” autism is meant specifically an autism syndrome thatappears in a child (generally between 12 and 18 months old) who haspreviously been developing normally. Symptoms include loss of language,social, and play skills, and onset of autistic characteristics such asavoidance of eye contact, self-stimulation behaviors, etc. Other formsof autism are clinically distinct in onset, for example early onsetautism, where affected children may be born with the autistic conditionor it may develop very early in life. Conventional theories are thatthere are genetic underpinnings to early onset autism and exposure toxenobiotics that lead to defects in normal immunity, but it is morelikely in at least some cases that there is a gastrointestinalcomponent, for example, infection with toxin-producing organisms becauseof a not yet fully developed normal flora (as in infant-botulism).

Eleven children with regressive onset autism were recruited for anintervention trial using a minimally absorbed oral antibiotic. Entrycriteria included antecedent broad-spectrum antimicrobial exposure,followed by chronic persistent diarrhea, deterioration of previouslyacquired skills, and then autistic features. Short-term improvement wasnoted using multiple pre-and post-therapy evaluations. These includedcoded, paired videotapes scored by a clinical psychologist blinded totreatment status which noted improvement in 8 of 10 children studied.Unfortunately, these gains largely waned at follow-up. Although theprotocol utilized is not suggested as useful therapy, these resultsindicate that study of a possible “gut-flora” connection warrantsfurther investigation as it might lead to greater pathophysiologicinsight and meaningful prevention and/or treatment in a subset ofchildren with autism.

Autism is a devastating and largely untreatable disorder currentlyclassified as a Pervasive Developmental Disorder in the DSM-IV, itusually manifests in early infancy, with impairment typically persistinginto adulthood. Recent incidence estimates are one in 110 children(˜10%) (CDC) with males four times more likely to be affected. Althoughsome children are later found to have chromosomal aberrations ormetabolic disorders which may explain their autistic features, nounderlying etiology can be identified in the vast majority of cases.“Autistic regression” occurs in approximately one third of cases, withregression typically occurring before two years of age, and involvingloss of language, social, and play skills.

Hypothesis

A number of parents of children with regressive onset autism reported tous their observation of the following sequence: repeated broad-spectrumantimicrobial use (usually for chronic otitis media), followed bychronic diarrhea, then loss of language, play, and social skills, andsubsequent onset of autistic symptoms. We developed the hypothesis thatrepeated antimicrobial use may have disrupted a protective effect ofindigenous intestinal organisms and allowed colonization by one or moreneurotoxin-producing species. If this were true, then appropriatelytargeted antimicrobial therapy might reduce autistic symptoms in theseindividuals. The most plausible candidate organisms appeared to be oneor more clostridial species. We now know that Desulfovibrio species arekey organisms in autism.

Treatment Rationale

Therapeutic options include metronidazole, bacitracin, or vancomycin.The latter was chosen for its efficacy, minimal absorption (i.e., theantibiotic remains in the intestinal tract and is excreted in thestool), and benign taste (the unpleasant tasting metronidazole orbacitracin would have required a nasogastric tube for drug delivery).The decision to use vancomycin was not made lightly, however, since thisdrug is of paramount importance in treating life-threateningantibiotic-resistant bacterial infections, and significant public healthconcerns exist should its use become widespread in the community. It wasused only to establish the point that autism is related to certainintestinal bacteria and that significant improvement can occur withelimination of the suspect bacteria.

Index Case

The index case was a 4.5 year old Caucasian male with chronic diarrheaand autism whose motor, cognitive, and social development was normaluntil 18 months of age. Diarrhea began at approximately 17 months of ageafter three 10 day courses of broad spectrum antimicrobials prescribedover a six week period for “chronic otitis media.” There was no blood orpus in the stool nor associated constitutional symptoms. At 19 months ofage there was profound behavioral and developmental deterioration, alongwith emergence of severe autistic features.

Extensive genetic, neurologic, gastrointestinal, and immunologicevaluations were all unrevealing. Neither conventional (e.g., full-dayspecial education program, speech and play therapy) nor unconventionalinterventions (e.g., special diets, megavitamin loading) had asignificant effect on his autistic symptoms.

A 12 week therapeutic trial of oral vancomycin (125 mg QID) was begunwith expanded observations by a pediatric neuropsychologist pre-andpost-treatment. At baseline, the child was not on a special diet nor washe taking any vitamin supplements. Three days after initiation of thevancomycin therapy, a hyperactivity pattern emerged which lasted forfour days. This was followed by two days of lethargy, and subsequentlyby a rapid and dramatic clinical improvement. He became affectionate andrelatively calm. He promptly achieved toilet training and increasedvocabulary. Follow-up behavioral observations after eight weeks oftherapy noted an increase in on-task performance, compliance withparental requests, awareness of environmental surroundings, andpersistence when engaging in positive activities. A significantreduction in repetitive and self-stimulatory behaviors was also noted.The child's educational therapies remained unchanged for both six monthsbefore and during the vancomycin trial. Shortly after vancomycindiscontinuation, behavioral deterioration was observed. Though stillimproved over baseline, he eventually lost most of the initial gains.

Methods Subjects and Study Design

To explore whether our index case's improvement represented a truetherapeutic effect, institutional human investigation committee approvalwas obtained for an open-label trial in a narrowly defined subgroup ofautistic children. Eleven children (10 males, 1 female; age range: 43-84months) were enrolled. Inclusion criteria for the study were derivedfrom our central hypothesis and index case characteristics. Theyinclude 1) Meets diagnostic criteria for Autistic Disorder (DSM IV299.00); 2) Other genetic and medical diagnoses have been adequatelyevaluated and ruled out; 3) Definable, rapid onset after 12 months ofage; 4) Antecedent antimicrobial use (≦2 months of autism symptomonset); 5) Persistent loose stool history, with diarrhea onset beforeautism symptoms; 6) Symptoms for ≦4 years; 7) Child is 2-8 years of age;8) No evidence of any significant medical problem that might complicatetreatment such as renal, cardiac or pulmonary disease, severeenterocolitis (visible blood or pus in the stool), or chronic infection(e.g., tuberculosis); and 9) Clinically static for 3 months (no newneuroleptic, seizure, or other medications), with no elective changesduring the study, and 10) No antimicrobial use for at least 2 monthsprior to entry into the study. All children had diarrhea and regressiveonset of autistic features (occurring at a mean of 17.7±3.4 months) aspreviously defined in the literature.

The Developmental Profile II provided descriptive developmental levelsto contrast with developmental age. While mean chronological age of thechildren was 59.4±12.7 months, the mean developmental age for thedomains of communication (23.0 months±13.0), socialization (25.6months±12.9), and self-help (34.4±12.4) are evidence of theirsignificant developmental delay. The Childhood Autism Rating Scale(CARS) was also administered. The CARS is a 15-item behavioral ratingscale developed to identify children with autism, and to distinguishthem from developmentally handicapped children without the autismsyndrome. Based upon CARS diagnostic categories, six children met thecriteria for severe autism, two for moderate autism, and three for mildautism. The vancomycin dose was 500 mg/day given orally as a liquid (500mg/6 ml), divided into 2 ml TID for eight weeks. This was followed byfour weeks of oral treatment with a probiotic mixture of Lactobacillusacidophilus, L. bulgaricus, and Bifidobacterium bifidum (40×10⁹ cfu/ml).

Psychological Evaluations

Two measures of potential improvement were examined: I) Children werevideotaped for 30 minutes at baseline and once during therapy in aplayroom environment. At each session, the child was directed to playwith a series of puzzles, books, blocks, and dolls by the mother andthen by the evaluator. At the end of the trial, a clinical childpsychologist (who was provided with a brief explanation of our workinghypothesis) compared coded, paired videotapes of 10 of the 11 childrenstudied (video was not available for one child). The psychologist viewedeach pair of tapes and scored them. To diminish the possibility ofinvestigator bias, the tapes were randomly numbered and the psychologistdid not have any personal contact with the children. 2) Behavior andcommunication analog rating scales were completed by the study physicianat baseline, during therapy, and at follow-up in a manner similar topreviously validated methods for other disease states. Results arepresented as median scores to account for potential non-linear scoreincrement.

Laboratory Evaluations

Extensive medical evaluations were conducted in parallel with thedetailed psychological assessments. Stools were examined for occultblood, inflammatory cells, Aeromonas hydrophila, Cryptosporidium,Clostridium difficile toxin, routine bacterial pathogens, and ova andparasites. Blood tests included complete blood cell counts, chemistrypanels, and erythrocyte sedimentation rates. Urinalyses were alsoobtained. Detailed quantitative aerobic and anaerobic fecalmicrobiologic studies were conducted at the Wadsworth AnaerobicBacteriology Laboratory on specimens from four children. Each stool wascultured with a total of 27 different media and atmospheric conditions,modified from the procedure described in Summanen et al.

Results Analog Rating Scales, Videotapes, Treatment Observations andLaboratory Evaluations

Unblinded assessment using an analog rating scale noted improvement forthe group as a whole in communication (Wilcoxon Signed Ranks Z=2.9,p=0.003) and behavior (Wilcoxon Signed Ranks Z=−2.9, p=0.003). To insurethat changes attributed to intervention were not a reflection ofdifferences at baseline, Spearman correlations were conducted. Therewere no significant correlations between the baseline measure andpost-intervention score for either communication (rho=0.35, p=0.28) orbehavior (rho=0.22, p=0.51). Blinded assessment of the coded, pairedvideotapes noted an improvement during therapy in eight of ten childrenstudied, no change in one, and a possible deterioration in one.

As previously observed in the index case, a brief (1-4 days) period ofhyperactivity was noted in six children within three days of initiatingantibiotic treatment. One subject then experienced a day of markedlethargy. Otherwise, aside from obvious autistic features, all childrenhad normal physical examinations at baseline and throughout the study,as well as unremarkable basic blood, stool, and urine tests as outlinedin the Methods section.

Long-Term Follow-Up

Although apparent improvement was clear by several measures,unfortunately these gains did not endure. One child who had respondedsignificantly to treatment, deteriorated towards the end of the studywhile still on vancomycin therapy. During telephone follow-up (conductedweekly during the probiotic therapy), most parents reported substantialbehavioral deterioration within two weeks of discontinuance ofvancomycin treatment. Due to difficulty in disguising the taste,probiotic treatment compliance was very poor in several children.Behavioral deterioration appeared to occur whether or not the child wascompliant with the probiotic therapy regimen. Therefore, it would appearthat the probiotic therapy used as an adjunct after vancomycin treatmenthad no discernible beneficial or adverse effect. All children wereobserved in follow-up, ranging from two to eight months afterdiscontinuance of vancomycin. In all but one child, the analog ratingsreturned towards baseline.

Quantitative Fecal Flora

Given the extreme labor intensiveness of such studies, it will be sometime before detailed microbiologic analysis of all pre-and post-therapystool specimens is completed. Stool specimen data from four autisticchildren prior to vancomycin therapy were compared to those of 104normal adult subjects from previously published studies (performed underthe supervision of the same principal investigator). Anaerobic cocci,chiefly peptostreptococcal species, were, present in 93% of the adults'specimens, comprising some 10% of the stool microorganisms. In starkdistinction, these species were absent from the stools of each of thefour autistic children tested (Table 3).

TABLE 3 Fecal Flora Data Autistic Autistic Autistic Autistic AdultsOrganism Patient A Patient B Patient C Patient D (104 Subjects*)Enterobacteriaceae 6 7 7 7  9 Streptococcus 3 5 0 4  9 Enterococcus 0 60 0  8 Bacteroides fragilis grp 8 8 9 8 11 Bacteroides, other 8 0 9 8 11Anaerobic GNR, other 6 4 7 5  8 Peptostreptococcus spp. 0 0 0 0  10**Anaerobic cocci, other   0*** 0   0**** 0  11** Lactobacillus spp. 9 910  8 10 Bifidobacterium spp. 7 9 9 8 10 Eubacterium spp. 8 0 9 8 11Clostridium spp. 9 7 8 8 10 Units are log₁₀ colony forming units (cfu)gram dry weight. *Mean of positive specimens. Subjects were normaladults on various diets (vegetarian, traditional Japanese diet, orstandard Western diet); there were no statistically significantdifferences in the results between these various groups. **93% of the104 subjects had Peptostreptococcus spp. and/or other anaerobic cocci.***Ethanol and heat-resistant coccoid forms were present (probablyclostridia.) ****Heat-resistant coccoid forms were present (probablyclostridia.)

Discussion

The apparent, though short-term, improvement during treatment with thisminimally absorbed antibiotic is not explainable using currentconventional genetic hypotheses' alone for autism. Results of thispreliminary study, along with previous reports of increased intestinalpermeability and a “nonspecific colitis” in children with autism,suggests a possible “gut-brain” etiologic connection may be present in asubset of these children.

Although the hypothesis that autism (in a defined subset of children)may be a sequela to the colonization of the intestinal tract by one ormore neurotoxin-producing bacteria is novel, published data alongseveral paths may lend credence to the notion that an alteration incolonic flora contributes to autism symptoms. The first line of evidenceis from the infant botulism literature. This condition was firstrecognized as a distinct clinical entity in 1976. It differs fromclassical (foodborne) botulism in that the intestinal tract becomescolonized by Clostridium botulinum and elaboration of the neurotoxinoccurs in vivo. Age is a primary risk factor for the development ofinfant botulism as diagnosis of the disease is rare after 1 year of age.Studies in animals have demonstrated a similar age-dependentsusceptibility. However, the colonization resistance observed in matureanimals is greatly diminished when they are treated with broad-spectrumantimicrobials. Similarly, antimicrobial use has been identified as arisk factor for the development of botulism related to intestinalcolonization with C. botulinum in older children and adults.

The second line of evidence is from human and animal studies which haverepeatedly demonstrated that intestinal colonization by opportunisticpathogens (e.g., Escherichia coli, Klebsiella pneumoniae, Pseudomonasaerguinosa, Salmonella enteritidis, Shigella flexneri, and Vibriocholerae) is greatly enhanced when protective intestinal microbiota isdisrupted by broad-spectrum antimicrobials. In humans, thebest-documented example of opportunistic colonization of the intestinaltract following antimicrobial use is that by Clostridium difficile, thecausative agent of pseudomembranous colitis.

Another potentially relevant condition is d-lactic acidosis, in whichassociated psychiatric symptoms are well-documented. D-lactic acidosis,a complication of short bowel syndrome or intestinal bypass surgery forobesity, is a condition caused by a change in bacterial flora to anacid-tolerant, aciduric (Lactobacillus, Bifidobacterium, Eubacterium,and Streptococcus) flora. Patients present with a range of behavioralchanges such as hostility, slurred speech, stupor, altered mentalstatus, dizziness, asterixis, and ataxia. Treatment is with appropriateoral antimicrobials, resulting in rapid cessation of neurological signs.

For quantitative measurement of improvement in autistic symptomotology,the current study utilized two independent assessment tools. Althoughthe analog rating scales were completed by the study physician who wasaware of the children's treatment status, the formal videotape ratingswere performed in a blinded manner. The improvement observed aftervancomycin intervention appeared to be significantly greater than couldnormally be attributable to the characteristic waxing and waning ofautistic symptomotology.

A substantial deterioration of the behavioral improvements made while ontherapy was reported by most parents within two weeks of ending thevancomycin trial. While the cause for neither the apparent improvementnor the later decline is known, it is possible the deterioration is dueto the offending organism being spore-forming, and hence survivingtherapy to germinate after vancomycin discontinuation, as has beendocumented with Clostridium difficile infection. An additionalpossibility is that the therapy was sublethal due to antimicrobialchoice and/or dosage regimen permitting emergence ofantimicrobial-resistant bacteria.

Since vancomycin is not absorbed, it appears likely that the behavioralimprovement was related, in some way, to the drug's effect on theintestinal tract flora (and not a “drug effect” per se on the centralnervous system). Although we theorize that the transient benefit fromvancomycin treatment may be due to the temporary elimination of aneurotoxin-producing pathogen, there are other possible mechanisms. Forexample, autoantibodies to neuron-axon filament protein, glialfibrillary acidic protein, and myelin basic protein have been reportedin autism and it has been postulated that these autoantibodies maycontribute to autistic symptomotology. It is at least theoreticallypossible that the production of these autoantibodies is related to thepresence of an infectious pathogen as has been postulated for rheumatoidarthritis.

The significance of the possible fecal flora changes in these autisticchildren is unknown. It is unlikely that specimen collection or shippingcontributed to the absence of Peptostreptococcus and other anaerobiccocci as other equally oxygen-sensitive organisms were recovered.Although all of the children had previously received broad-spectrumantimicrobials (capable of severely disrupting intestinal flora), fecalbacterial counts typically return to their pre-treatment compositionwithin two weeks of discontinuance of the antimicrobial agent.^(iv)Therefore, since none of the children, at base line, had a history ofantimicrobial treatment for at least two months prior to entering ourstudy, it is unlikely that the absence of these species reflects atransient alteration in the children's fecal flora. An uncharacterizedPeptostreptococcus species has been documented to inhibit certainorganisms, including clostridia, in vitro and in animals, and it isintriguing to speculate that the absence of such organisms in certainautistic children may permit growth of clostridial or othertoxin-producing bacteria through loss of competitive inhibition.

The fecal flora of pediatric subjects has been extensively studied. Useof normal adult control fecal specimens in the present study, though notideal, is justifiable given documented similarity to pediatric stoolflora. For example, one recent review of bacterial colonization patternsstates that “by 12 months (of age) the anaerobic fecal populations beginto resemble that of adults in number and composition as the facultativeanaerobes decrease. By two years of age, the profile resembles that ofthe adult.”

Example 2 Culture Conditions, Antimicrobial Susceptibility DeterminationCulture Conditions

We use a selective medium for clostridia that contains (per liter) 25.0g of brain heart infusion (BBL, USA), 20.0 g of agar (Sigma, USA), 76.0mg of sulfamethoxazole, 4.0 mg of trimetoprim, 1.0 mg of vitamin K, 5.0mg of hemin, and 50.0 ml of laked sheep blood. All medium componentsexcept the two antimicrobial agents and the laked sheep blood are mixed,autoclaved at 121° C. for 15 mins and cooled to 50° C. in a water bath,at which point the three initially omitted ingredients are added. Anadditional medium is made up in identical fashion except that 30.0 to50.0 g of agar is used, rather than 20.0, in order to make the mediumstiffer and thus minimize spreading of clostridial colonies.

Stock solutions of antimicrobials are prepared separately in advance byaseptically dissolving the sulfamethoxazole in half volume hot waterwith a minimal amount of 2.5 M NaOH and the trimetoprim in 0.05 N lacticacid or HCl, 10% of final volume. The stock solutions are stored at −20°C. before addition to the selective medium. After the medium is pouredinto Petri dishes, the plates are dried and placed into an anaerobicchamber and reduced for approximately 24 hours. They are then stored inthe chamber at ambient temperature (25° C.) for at least two days, butno longer than seven days, before use.

The entire stool specimen is weighed before processing. It is thenplaced into an anaerobic chamber and homogenized in a heavy duty blenderwith no diluent (if liquid) or with one or two volumes of diluent (0.05%yeast extract) added if the stool is soft or fully formed.Homogenization is carried out because we have found previously thatorganisms are not distributed evenly throughout the fecal mass; thisavoids sampling errors. Serial ten-fold dilutions of the specimen arethen made in 9 ml dilution blanks (Anaerobe Systems, USA) and 100 μl ofeach dilution from 10⁻¹ through 10⁻⁸ is inoculated onto the selectivemedium (both agar concentrations) and onto a Brucella blood agar plate.The fecal suspensions (10⁻¹-1⁻⁵) are also heated at 80° C. for 10minutes (to select out clostridial spores) and 100 μl of each dilutionis inoculated onto the selective media and the Brucella blood agar.

After 5 days of incubation of the inoculated plates at 37° C., eachcolony type from both heat-treated and non-treated specimens is countedfrom a dilution plate containing between 30 and 300 colonies of the typebeing isolated. Total bacterial counts, in addition to clostridialcounts, are also recorded from the Brucella blood agar plates.

In order to correct for differing moisture content in differentspecimens of stool, a portion of sample (˜1 g) is placed onto apre-weighed drying dish. The dish is again weighed and then placed intoa drying oven and incubated at 70° C. (with 18-20 inch Hg vacuum) for 48hours. After this incubation, the dish with the specimen is re-weighedso that bacterial counts can be corrected for moisture content.

Identification of Isolated Bacteria

The identification of isolated colonies as clostridia, and speciation ofthese, is done by methods outlined in the Wadsworth AnaerobicBacteriology Manual, 5th Edition (Summanen et al., Star Publ. Co.,Belmont, Calif., 1993, herein incorporated by reference) including, whenindicated, cellular fatty acid analysis in a MIDI capillary column gaschromatograph, 16S rDNA sequencing, and DNA-DNA hybridization (thelatter two procedures as outlined in a paper from this laboratory(Wexler H M et al., Int. J Syst Bacteriol 46:252-258, 1996, hereinincorporated by reference).

Antimicrobial Susceptibility Determination

Testing of susceptibility of isolated clostridia to antimicrobial agentssuch as vancomycin, metronidazole, bacitracin and ramoplanin is done bytwo different techniques—the NCCLS Wadsworth agar dilution procedure(Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria,Approved Standard-Fourth Edition. NCCLS Publication M 11-A4, Wayne, Pa.:NCCLS, 1997, Vol. 17, No. 22, all herein incorporated by reference) andthe spiral gradient endpoint procedure (Wexler H M et al., J ClinMicrobial 34:170-174, 1996, herein incorporated by reference).

Example 3 Testing for Toxin Polypeptides ELISA Testing—Rationale andMethods

Since all of the known clostridial neurotoxins share significant aminoacid homology, low-level cross-reactivity of antibodies has beenreported. This will allow us to detect a clostridial neurotoxin that isclosely related to, but not identical with, tetanus toxin.

Media containing hydrolysates of casein improve the production of allknown clostridial neurotoxins. Therefore, the cells were grown in BrainHeart Infusion Broth (Becton Dickinson, 20 Sparks, Md.) supplementedwith 2.5% pancreatic digest of casein (Tryptone Peptone, BectonDickinson). After five days of growth, the culture supernatants wereclarified by centrifugation at 4000×g and filter-sterilized through a0.45 μl nitrocellulose membrane filter. Antigens from known C. tetanistrains (ATCC 10779, 19406, 453, 9441) and tetanus toxoid (Lederle,Pearl River, N.Y.) were used for initial optimization experiments andsubsequently as positive controls.

Our methods are based upon previously standardized ELISA protocols fordirect competitive detection of soluble antigens (Current Protocols inMolecular Microbiology). The wells of solid-phase immunoassay microliterplates (Biotech Diagnostic, Niguel, Calif.) are inoculated with 50 μl ofantigen solution, sealed with plastic wrap and incubated overnight atroom temperature. The plates are washed three times with deionized waterto remove unbound antigen solution. The wells are then filled with ablocking buffer (Tween 20 0.05% and bovine serum albumin 0.25%) andincubated at room temperature for 30 minutes. The plates are againwashed three times prior to addition of 50 μl of serially dilutedantibody solution; 1:1000 to 1:10,000 dilutions of polyclonal IgG goattetanus exotoxin (Fitzgerald, Concord, Mass.). Plates are sealed withplastic wrap and incubated at room temperature for ⊃ two hours. Afterwashing, rabbit anti-goat IgG alkaline phosphatase conjugated antibodies(Fitzgerald) are added and the plates incubated at room temperatureovernight. A microtiter plate reader was used to measure thefluorescence.

ELISA Results

All four ATCC strains of C. tetani consistently produced positiveresults. This is interesting to note because C. tetani strain ATCC 19406does not consistently yield positive PCR results. One possibleexplanation may be that ATCC 19406 produces a toxin immunologicallysimilar (or identical) to other C. tetani strains but its genetic codefor toxin production is slightly different

During initial testing, we noticed that all C. perfringens strains (ATCCtype strain, strains from children with autism, and strains from normalchildren) yielded positive results. This might be due tocross-reactivity of the antibodies against tetanolysin (a hemolysinproduced by C. tetani strains) with perfringolysin—a very closelyrelated hemolysin. We performed Western blot testing so that the size ofthe immunoreactive proteins could be visualized and compared to positivecontrols.

Western Blot Testing

The cells were grown in Brain-Heart-Infusion Broth (Becton Dickinson,Sparks, Md.) supplemented with 2.5% pancreatic digest of casein(Tryptone Peptone, Becton Dickinson). After four days of incubation at37° C. and an additional two days at 4° C. (to enhance sporulation,lysis and release of toxin), the culture supernatants are clarified bycentrifugation at 4000×g and filter-sterilized through a 0.45 μmnitrocellulose membrane filter. Clostridium tetani strains (ATCC 10779,19406, 453, 9441) and tetanus toxoid (Lederle, Pearl River, N.Y.) wereused for initial optimization experiments.

Our methods are based upon previously standardized protocols forimmunoblotting and immune detection (Western blotting) of solubleantigens (Current Protocols in Molecular Microbiology, vol. 2, 1997, pp.10.8.1-21). Briefly, the filtered culture supernatant is solubilizedwith a detergent (SDS) and a reducing agent is included to reducesulfhydryl bonds. The solubilized proteins are separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel is thenelectroblotted resulting in transfer of the protein bands to anitrocellulose membrane. The membrane is placed in a tray with blockingbuffer, 2% skim milk in phosphate-buffered saline (PBS), and kept atroom temperature for 1 hour. Primary antibody, polyclonal IgG goattetanus antitoxin (Fitzgerald, Concord, Mass.), diluted 1:1,000 inblocking buffer is then added. Following a 1-hour incubation, themembrane is washed four times with PBS. The detection of antibodybinding occurs with rabbit anti-goat-IgG conjugated to alkalinephosphatase. When substrate is added, a calorimetric reaction occurs,thus indicating that the initial (anti-tetanus serum) antibody was boundby a protein on the membrane. (Sigma, St. Louis, Mo.) Anti-Ig conjugate,1:1,000 dilution in blocking buffer, is added and incubated at roomtemperature for 1 hour. After four fifteen-minute washes, the membraneis incubated with color development buffer (100 mg/ml 4-nitro bluetetrazoliurn chloride (final: 0.33 mg/ml) (NBT) and 50/mg/ml5-bromo-4-chloro-3-indolyl-phosphate (final: 0.165 mg/ml) (BCIP) addedto substrate buffer: 0.05M Na₂CO₃, 0.5 mM MgCl₂ pH 10.2). The reactionis stopped by washing the membrane in distilled water for 10 minutes.

Western Blot Results

We initially tested multiple C. perfringens strains; the ATCC typestrain, a strain isolated from the stool of a child with autism, and astrain isolated from the stool of a normal child. All strains of C.perfringens produced an immunoreactive protein of the same molecularweight, which explains the positive results observed during ELISAtesting. We theorize that this protein may be perfringolysin (whichwould be expected to cross react with anti-tetanolysin antibodies).There were, however, striking differences between these three C.perfringens strains. The strain from the autistic child producedadditional immunoreactive proteins. Furthermore, these immunoreactiveproteins appeared to be of the same molecular weight as the tetanusneurotoxin proteins (light and heavy chain, about 100 kD) produced byour C. tetani control strain. Repeat testing confirmed our initialresults. Additional studies were performed on several clostridialspecies isolated from a second child with autism. One of the strainsfrom this child, C. beijerinckii, produced a strongly immunoreactiveprotein of ˜50 kDa, which is the approximate weight of the light-chainof tetanus toxin and other known clostridial neurotoxins. Western blottesting of the ATCC C. beijerinckii type strain will be performed.However, ELISA testing of the type strain was negative, suggesting thattypical C. beijerinckii strains do not produce a protein that isimmunoreactive with anti-tetanus antibodies.

Our colleague, has tested the filtrate of an ATCC strain of C. tetani inthe hind leg of mice and has produced paralysis of that limb andsubsequently death. We will test blinded cultures from autistic andcontrol children for this in vivo test.

Example 4 Fecal Microflora of Autistic Children Materials and Methods

Subjects. 33 autistic subjects, 7 non-autistic siblings and 8 controlsubjects considered here were enrolled in the study at the EvergreenCenter in Oregon City, Oreg. and the Center for Autism and RelatedDisorders (CARD) in Tarzana, Calif. with signed, informed consent oftheir parents or guardians and approval of VA Greater Los AngelesHealthcare System Institutional Review Board IRB B. All autisticchildren participating in this study had an educational or developmentalpediatrician evaluation and were diagnosed with autistic spectrumdisorder. Subsequently, JAG evaluated each patient for autism andvalidated the diagnosis based on impairment in social skills, impairmentin language skills and verbal communication, sensory disturbances,repetitive stereotypical behaviors, and gastrointestinal disturbances.JAG's practice has been limited to autistic spectrum disorders since1999 and he has evaluated and treated approximately 2000 patients inthat time. All autistic and control subjects were between 2 and 13 yearsof age. Among the autistic group, there were 24 males and nine females.Among the control group, there were five males and three females. Thesibling control group consisted of five females, and two males. Basedupon clinical evaluation we were able to distinguish the severity ofautism in 30 of the 33 autistic subjects. Eleven subjects werecategorized as severe, while 19 subjects were grouped into the mildlyautistic category.

Stool collection and transport. Specimens (the entire fecal specimen)were collected at the homes of the participants and were shipped to theWadsworth Anaerobe Laboratory in Los Angeles the same day, by airexpress, packed with frozen blue shipping packets. The specimens alwaysarrived in Los Angeles the next morning. In the laboratory, thespecimens were thawed, homogenized in a Waring blender, and DNA wasextracted. The DNA specimens were then kept frozen at −80° C. forpossible further studies.

DNA extraction. One ml aliquots of stool, previously diluted 1:3 insterile bi-distilled water and thoroughly homogenized under anaerobicconditions in an anaerobic chamber) were centrifuged at 14,000×g for 3min to pellet fecal bacterial cells. The supernatant was carefullyremoved and discarded. Two hundred milligrams of cell pellet wastransferred to a fresh tube and subjected to DNA extraction using acommercial extraction system (QIAamp DNA stool mini kit; Qiagen)according to the instructions of the manufacturer. Previous studies inour laboratory have shown that the QIAamp product produces high-qualityDNA free of PCR-inhibiting substances. DNA extraction was performed induplicate.

Blinding of information. Information on which specimens came fromautistic children and which from control children was withheld from theinvestigators doing the pyrosequencing until their data set wascompleted. Subsequently, this information was provided to them by thegroup in Oregon so that proper group analyses of the data could becompleted.

bTEFAP. bTEFAP was utilized with titanium chemistry, as describedpreviously by the Dowd laboratory (Dowd et al. PLoS ONE 3, e3326, 2008;Dowd et al, Foodborne Pathog. Dis. 5, 459-472, 2008; and Dowd et al. BMCMicrobiol. 8, 125, 2008; which are incorporated herein by reference), toevaluate the bacterial populations in the feces of separate autisticchildren in comparison to individual control samples. In preparation forFLX sequencing (Roche, Nutley, N.J.), the DNA fragments' size andconcentration were accurately measured. A small sample ofdouble-stranded DNA molecules/μl was combined with DNA capture beads,and then amplified by emulsion PCR. After bead recovery and beadenrichment, the bead-attached DNAs were denatured with NaOH, andsequencing primers were annealed. A 454 sequencing run was performedusing the Genome Sequencer FLX Titanium System (Roche, Nutley, N.J.).All FLX-Titanium procedures were performed using Genome Sequencer FLXSystem manufacturer's instructions (Roche, Nutley, N.J.).

bTEFAP sequence processing pipeline. Custom software written in C#within a Microsoft®.NET (Microsoft Corp, Seattle, Wash.) developmentenvironment was used for all post-sequencing processing. Quality trimmedsequences obtained from the FLX Titanium sequencing run were deriveddirectly from FLX Titanium sequencing run output files. Tags wereextracted from the multi-FASTA file into individual sample-specificfiles based upon the tag sequence. Sequences which were less than 350base pairs after quality trimming were not considered. Sequences wereanalyzed by a script optimized for high throughput data. Definitechimeras were removed using B2C2 (software are available athttp://researchandtesting.com/B2C2). The resulting FASTA for eachsample, with chimeras removed, were then evaluated using BLASTn againsta custom database derived from NCBI, curated based upon quality criteriasimilar to that utilized for high quality sequences of the RDP-IIdatabase. C# scripts were used to extract necessary taxonomicinformation from NCBI for the accession numbers derived from thedatabase queries.

Microbial diversity analysis was performed by clustering sequence tagsinto groups of defined sequence variation ranging from unique sequences(no variation) to 10% divergence evaluated, as previously described,from raw reads of comparable Phred20 quality (>350 bp) (Acosta-Martineset al., Soil. Biol. Biochem. 40:2762-2770, 2008, which is incorporatedherein by reference). Clusters acting as OTUs were used to generaterarefaction curves and as input for calculations with theabundance-based coverage estimator ACE and the Chao1 (Chao et al.,Biometrics 58, 531-539, 2002, which is incorporated herein by reference)estimator of species diversity. Table 7 shows the microbial diversityestimate averages and t-test results obtained with (parametric andnon-parametric) modeling of rarefaction, ACE and Chao 1. Final datasetsclassified at the species and other relevant taxonomy levels werecompiled into separate worksheets. To assess not only the overallbacterial richness of the samples, but the actual populations, weconducted a “composition analysis”. This process produced resultscontaining information for each sample at each taxonomic level (kingdom,phylum, class . . . ).

Statistics. Principal Component Analysis

To assess the separability of the samples, Principal Component Analysiswas implemented. Principal Component Analysis (PCA) (HSPH. Autism HasHigh Costs to U.S. Society. Harvard School of Public Health. (2006)http://www.hsph.harvard.edu/news/press-releases/2006-releases/press04252006.html)is widely used for dimensionality reduction to help with visualizationof high dimensional data. PCA is defined as the orthogonal projection ofthe data onto two or three dimensional space such that the variance ofthe projected data is maximized. Custom Python scripts tailored for nextgeneration data (distance matrices and taxonomic abundance) wereimplemented to assess bacterial composition of samples and determine the3 Principal Components. This data is visualized by plotting the sampleson axes defined by the principal components. Samples more similar toeach other should appear closer together according to the respectiveaxis reflecting the variation among all samples. This technique isuseful in displaying clusters existing within data. The variables(features) are the relative bacterial composition in a sample at aparticular taxonomic level.

Clustering

To analyze the relationships and clustering between autistic and controlsamples, double dendrograms were formed based on the bacteriacomposition information. The analysis was performed using the NCSSStatistical Software as described previously (Acosta-Martines et al. 4Soil. Biol. Biochem 40, 2762-2770, 2008; Bailey et al. Biol. Lett. ePubahead of print, 2010; Dowd et al. Foodborne. Pathog. Dis. 5, 459-472,2008; Suchodolski et al. BMC Microbiol. 9, 210, 2009; and Wolcott et al.BMC Microbial. 9, 226, 2009; which are incorporated herein byreference).

Other Statistics

As appropriate, student's t-tests were used for comparing means withinvarious groups of data.

Results and Discussion

The results indicate there is a significantly higher diversity ofbacteria found in the feces of autistic subjects compared to controls(Table 4).

TABLE 4 Diversity and richness data for groups of subjects in the study.Data are presented at the 1% divergence level (corresponding roughly tothe strain of bacteria), the 3% divergence level (corresponding roughlyto the species level) and the 5% divergence level (corresponding roughlyto the genus level) for rarefaction maximum predicted (RFM), ACE, andChao1 estimates. The P-values (p-val) corresponding to a T-testevaluation, indicate that the controls have significantly lower numbersof operational taxonomic units than the autistic subjects. RFM 1 RFM 3RFM 5 ACE 1 ACE 3 ACE 5 Chao1 1 Chao1 3 Chao1 5 Group Means Mild 886 558376 2627 1181 584 2265 1055 562 Autism Mean Mild 417 284 192 1519 680326 1298 607 329 Autism St. Dev Severe 914 564 375 2402 1122 567 21351052 546 Autism Mean Severe 240 150 107 665 330 192 583 297 186 AutismSt. Dev All Autism 871 542 364 2455 1118 561 2142 1018 541 Mean AllAutism 352 238 161 1250 565 274 1065 503 273 St. Dev Control 491 296 2091234 567 308 1092 530 300 Mean Control St. 64 66 39 462 209 78 318 16770 Dev Sib 1120 704 473 3032 1435 740 2694 1331 732 Control Mean Sib 319237 155 1079 529 256 883 461 259 Control St. Dev Group Student's T-testp values Severe vs 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.001control control vs 0.002 0.003 0.005 0.005 0.005 0.007 0.005 0.005 0.009all aut control vs 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000sib control sib vs 0.068 0.071 0.064 0.071 0.070 0.060 0.062 0.067 0.047severe aut

Even when relatively large genetic distances (5% divergence) areconsidered, these estimates predict that even at the genus level thereis significantly less diversity (at a 5% significance level) andrichness of microbial communities in control subjects than in theautistic group. These diversity results were similar when using threeseparate diversity and richness methods (including Chao 1, ace, andrarefaction). From this data we see that the parametric method ofrarefaction (Acosta-Martines 2008) predicts that the average number ofoperational taxonomic units present in the feces of all autistic samplesat 3% sequence divergence (the species level) was 542 compared to 296 inthe control samples. Using the ace non-parametric measure of richness,we see that there are a predicted 1118 species in the autistic samplesand 567 in the control samples and based upon Chao1 there were anaverage of 1018 and 530, respectively. This dramatic and significantlyincreased diversity and richness may be an important aspect of theautistic gastrointestinal microbiome. The increased microflora ofautistic children may contain harmful genera or species contributing tothe severity of autistic symptoms. Decreasing harmful populations withantibiotics like vancomycin has been shown to be an important step inimproving late onset autism symptoms.

The OTU data also indicates no statistically significant differencebetween the sibling control subjects and the severely autistic subjects.However, when comparing the true controls against sibling controls, theestimated richness does prove to be statistically different. This andother tests discussed later indicate the sibling controls to be moresimilar to autistic children than to the true control subjects.

Summary information at the phylum level for all four groups of samples(severely autistic, mildly autistic, control and sibling control) areshown in Table 5.

TABLE 5 Bacterial composition at the phylum level for control, siblingcontrol, mildly autistic and extremely autistic subjects (3 autisticchildren were not considered because of unknown severity). p-value Mild-Severe- Severe- Control S-Control Autism Autism Aut vs. Phylum (n = 8)(n = 7) (n = 19) (n = 11) Control Firmicutes 63.631 +/− 17.593 44.012+/− 24.576 38.975 +/− 15.434 38.015 +/− 13.772 0.001 Actinobacteria1.812 +/− 1.679 1.037 +/− 1.515 0.732 +/− 1.426 0.464 +/− 0.597 0.012Bacteroidetes 30.226 +/ 16.413   44.326 +/− 17.794 51.591 +/− 12.32751.248 +/− 7.043  0.001 Proteobacteria 0.535 +/− 0.428 2.327 +/− 3.7892.281 +/− 2.414 3.122 +/− 2.579 0.011 Verrucomicrobia 5.031 +/− 7.920 9.498 +/− 13.214 8.092 +/− 7.968  8.079 +/− 11.990 0.227 Cyanobacteria0.318 +/− 0.178 0.256 +/− 0.408 0.090 +/− 0.117 0.069 +/− 0.075 0.099Fusobacteria 0.081 +/− 0.0  0.0 0.024 +/− 0.010 0.024 +/− 0.0  0.194Tenericutes 0.0 0.110 +/− 0.079 0.789 +/− 0.117 0.167 +/− 0.209 0.098Lentisphaerae 0.0 0.0 0.037 +/− 0.0  0.0 0.0 The phylum designation isshown under the “Phylum” column. The next four columns display thepercentage at which the specified phylum can be found in a specificsample with a standard deviation proceeded with a “+/−”. Non-existingstandard deviations are designated with a 0.0. Control samples aredesignated under “Control”, sibling controls are in the “S-Control”column, levels for mildly autistic subjects are under “Mild-Autism”,while samples from the severely autistic are in the “Severe-Autism”column. Phyla not found in a group of samples are designated with a 0.0.The t-test based p-value is listed in the p-value column.

Bacteroidetes and Firmicutes are shown to be important phyla in thisanalysis. There are also significant differences between severelyautistic subjects and controls with regard to the Actinobacterium andProteobacterium phyla. A trend can be seen in Firmicutes where the levelof this phylum is much higher in the control than autistic samples.Taxonomically, it is not surprising at the phylum level thatBacteroidetes (Table 5) were significantly higher in counts in autisticsubjects (p 0.001) while Firmicutes tended to be higher in the controlsubjects (p 0.001). These data provide points to an altered microflorain the gut of autistic subjects. FIGS. 1A-1C show the composition ofautistic and control and sibling control samples, respectively, andagain emphasize the difference between the autistic and control groups.The sibling control figure (FIG. 1C), proportionally looks to be betweenthe autistic and control groups, as might be expected. However, similarto the autistic group, Firmicutes comprises less than 50% of thebacteria, unlike in the control group where Firmicutes represents anaverage of 63.6%.

Using the phylum composition data to analyze the microbiome further,Principal Component Analysis was performed. The analysis incorporates 9variables (the nine phyla represented in the samples). The datasuggested the autistic and control individuals were separable based onthe taxonomic percentages associated with each of the groups (FIG. 2A).FIG. 2A displays the mapping of all four classes at the phylum level andcovers 60.935 percent of the variation. The image accentuates thedifference of the control samples from the rest of the groups. While thecontrol points are more scattered across the grid, the mild and severeautistic points, along with the siblings of autistic children tend tocluster together. This can be more clearly seen in FIG. 2B where allautistic samples are grouped under the same color. This supports thesupposition that there is a difference in the fecal microflora betweenautistic and non-autistic children. Even children close to autisticindividuals seem to possibly be influenced by the bacteria, and overalldo not appear to be statistically different from children suffering fromautistic symptoms.

A similar graphical pattern of separation for the four groups propagatesthrough all taxonomic levels. In FIGS. 3A and 3B, the PCA results forthe genus level (198 variables/genera used for analysis) can be seen.Although the dividing line was less obvious and the three principalcomponents only covered 18.46% of the variation, the control samplesremained distinguishable from the rest of the samples.

Another observation associated with abundance data at the genus levelindicated that there are reduced populations of the Bifidobacteriumgenus in the severely autistic samples, compared with the controls.Evaluation of the Bifidobacterium genus-related data shows that therewas a significantly higher occurrence of species among the controlsubjects than in the autistic subjects (p<0.05) and though notindividually significant, the species were found at higher frequenciesin the control subjects (Table 6).

TABLE 6 Bifidobacterium spp. quantities and significance levels withinseverely autistic and control samples (non sibling). Avg A St. Avg C St.Species (n = 11) Dev A (n = 8) Dev C p-value B. adolescentis 0.125 0.2610.154 0.384 0.424 B. angulatum 0.000 0.000 0.046 0.089 0.050 B. animalis0.005 0.016 0.000 0.000 0.205 B. bifidum 0.012 0.020 0.017 0.048 0.372B. dentium 0.001 0.005 0.000 0.000 0.205 B. longum 0.084 0.150 0.6360.957 0.037 B. pseudocatenulatum 0.025 0.063 0.161 0.288 0.072 B.pseudolongum 0.000 0.000 0.012 0.034 0.126 B. saeculare 0.006 0.0200.000 0.000 0.205 Bifidobacterium genus 0.258 0.409 0.258Bifidobacterium species are listed in the “Species” column, along withthe average percentage at which they were found in the autistic andcontrol samples. These values are listed in the “Avg A” and “Avg C”columns for autistic and control samples, respectively, followed bytheir respective standard deviations listed in the “St. Dev” columns. At-test based p-value is listed under the “p-value” column.

Bifidobacterium along with Lactobacillus spp. are notable as probiotics,though little is known about strain specific differences, which may behost or individually specific (McCartney et al., Life Sci. 71,1893-1904, 2002). Probiotic therapy to alleviate symptoms ofgastrointestinal disorders has produced only slight or no improvement ofsuch disease states. The use of probiotics in autism-related disordershas also been discussed though little clinical evidence is apparent forthe efficacy of such treatment. Different species of Bifidobacteriumproduce different exopolysaccharides that act as fermentable substratesfor different human intestinal bacteria.

Other genera of interest are listed in Table 7.

TABLE 7 Top 20 occurring genera out of 198 of severely autistic andcontrol (non sibling) subjects. # A % Total # C % Total A, top 20 genera(n = 11) Flora A C, top 20 genera (n = 8) Flora C Bacteroides 11 35.544Bacteroides 8 24.481 Clostridium 11 10.343 Clostridium 8 17.748Faecalibacterium 11 10.173 Faecalibacterium 8 11.271 Eubacterium 115.521 Ruminococcus 8 7.581 Ruminococcus 11 3.329 Eubacterium 8 9.749Roseburia 11 2.033 Alistipes 8 2.621 Dorea 11 0.297 Roseburia 7 0.742Hespellia 11 0.176 Anaerofilum 5 0.104 Turicibacter 11 0.152Streptococcus 8 0.600 Akkermansia 10 7.344 Turicibacter 6 3.773Parabacteroides 10 5.222 Parabacteroides 7 1.980 Alistipes 10 4.296Dorea 8 3.504 Sporobacter 9 1.173 Veillonella 6 0.740 Bifidobacterium 90.258 Akkermansia 5 1.026 Anaerostipes 9 0.223 Sporobacter 1 0.054Ethanoligenens 9 0.113 Ethanoligenens 6 0.477 Anaerotruncus 9 0.092Papillibacter 5 0.140 Holdemania 9 0.084 Holdemania 6 0.107Phascolarctobacterium 8 1.382 Weissella 4 1.918 Desulfovibrio 8 0.276Dialister 3 0.032 The number of samples the bacterial species were seenin is listed in the # A or #C columns. The average percentage (% TotalFlora) designates the average percentage of the specific taxa found inthe total microflora in the group of samples containing the genus(autistic or control).

In Table 7, the top 20 genera, of a total of 198 encountered, indicateda similar overall composition between the severely autistic and controlgroups. However, Hespellia, Anaerostipes, and Desulfovibrio spp. wereseen in the top 20 genera only in the autistic subjects andStreptococcus, Veillonella, Weissella, and Papillibacter spp. were onlyin the control subjects among the top 20. The 19 genera with significantdifferences from this data include Turicibacter, Clostridium, Weissella,Parabacteroides and Ruminoccocus spp and others (Table 8).

TABLE 8 Significant genera among severely autistic vs non-siblingcontrol samples. # of # of Autistic Control avg % avg % p-val Genus (n =11) (n = 8) A C A vs C

0 6 0.000 0.095 <0.001

11 8 0.152 0.600 <0.001

11 8 10.343 17.748 0.001

8 8 0.240 1.228 0.005 Alkaliflexus 8 0 0.122 0.000 0.006Pseudoramibacter 5 5 0.027 0.132 0.011

8 3 0.276 0.032 0.011 Acetanaerobacterium 8 1 0.083 0.005 0.015

11 8 3.329 9.749 0.018

8 8 0.135 0.861 0.019 Anaerovorax 4 5 0.017 0.159 0.028

4 5 0.090 4.691 0.035 Lactococcus 0 3 0.000 0.028 0.035

10 7 5.222 1.980 0.036 Leuconostoc 4 3 0.010 0.052 0.040

9 6 0.113 0.477 0.041

11 8 35.544 24.481 0.044 Helcococcus 0 2 0.000 0.011 0.045 Alkaliphilus0 2 0.000 0.010 0.046 The number of samples of the specific bacterialgenus found is listed in the “# of Autistic” or “# of Control”,depending on which group the bacteria was found in exclusively. Theaverage percent of bacteria found are in the column “Avg % A” or “Avg %C” for autistic or control samples, respectively; “0.000” indicatesundetected. P-values are provided for comparisons of A vs C for each ofthe genera specified. A total of 198 genera were considered. Generalisted in bold are in the top 20 most predominant genera in extremelyautistic or control groups (Table 7).

The mean differences for the other 179 genera were not significant.Table 9 shows the various genera and species detected among theFirmicutes and Bacteroidetes phyla.

TABLE 9 Genera and species present in greater than 1% of the total florain one or more groups of children Autistic (No) Control (No) Mild SevereNormal Sibling (22) (11) (8) (7) % of the total flora Firmicutes:Clostridium aldenense 1.9 1.7 1.7 0.6 Clostridium hathewayi 2.3 1.6 1.21.6 Clostridium leptum 0.2 0.4 2.7 0.1 Clostridium methylpentosum 0.30.1 1.6 0.2 Clostridium orbiscindens 1.8 1.7 1.3 0.6 Dialister invisus0.6 0.1 4.7 1.1 Eubacterium eligens 1.9 3.0 0.6 1.7 Eubacteriumruminantium 0.2 0.1 1.7 1.4 Phascolarctobacterium faecium 1.5 1.4 1.92.2 Roseburia intestinalis 3.6 2.0 2.5 2.4 Sporobacter termitidis 1.01.2 0.7 1.5 Bacteroidetes: Alistipes onderdonkii 1.2 1.19 1.4 0.5Bacteroides caccae 1.7 0.5 4.9 0.9 Bacteroides stercoris 2.3 0.7 0.3 2.8Bacteroides vulgatus 13 12 3.6 2.5 Parabacteroides distasonis 2.5 2.51.6 1.1 Prevotella oulorum 0.2 2.0 0 8.6

Tables 10 and 11 show genera and species of possible importance incontributing to the clinical picture of autism or as protective flora,respectively.

TABLE 10 Genera and species of possible importance in contributing toautism % of Total Flora Severe Autism (11) Control (8) P ValueDesulfovibrio genus 0.28 0.03 0.010 Desulfovibrio piger 0.11 0.006 0.032Desulfovibrio desulfuricans 0.28 0 0.035 Desulfovibrio intestinalis 0.100.03 0.045 Bacteroides vulgatus 12.13 3.63 0.045

TABLE 11 Genera and species of possible importance as protective flora %of Total Flora Severe Autism (11) Control (8) P Value Collinsella genus0.02 0.62 0.050 Bifidobacterium genus 0.26 0.41 0.050 Bifidobacteriumlongum 0.08 0.64 0.037 Bifidobacterium angulatum 0 0.05 0.050 Dialisterinvisus 0.08 4.67 0.035 Clostridium leptum 0.35 2.70 0.010

Desulfovibrio was particularly interesting since all three species ofthis genus that were encountered showed significantly greaterpercentages of the total flora in the stools of severely autisticchildren than in controls. Furthermore, this sulfate-reducing genus hasbeen recovered from serious infections such as bacteremia. This genusproduces hydrogen sulfide, an important virulence factor, and is knownto corrode various metals. It might have the opportunity to attackcertain metals in the bowel.

Using the overall predominant genera, clustering analysis was performedto assess the importance of the bacterial gut flora of autistic childrenand the control and sibling control subjects. FIG. 4 shows the resultsof a clustering of all samples. Starting with the 198 genera, the numberof genera was reduced until there was a change in the clustering. Thusonly 27 of the 198 genera are needed to display the clustering patterns.The left and right sides of the double dendrogram show some indicationof grouping. In the left portion of the image, the mildly autisticsamples group together on the far left and other severity levels ofautism further to the right. The far right portion of the dendrogram isprimarily composed of the control and sibling control individuals. Basedon this information, there appears to be some indication of the gutmicroflora differing between the autistic and control groups. TheBacteroides genus, in particular, is an obvious indication of the changethat occurs from autistic to sibling and control children. The red color(bottom of FIG. 4), indicating a high abundance of the genus regressesto more orange and yellow tones (center of FIG. 4) indicating a decreasein the amount of the bacteria

Example 5 Materials and Methods Study Design

The study is limited to autistic children with late onset or regressiveautism and gastrointestinal (GI) abnormalities, notably abdominal painor discomfort, bloating, and constipation with or without diarrhea. Weused the stool samples from our pyrosequencing study¹², maintained at−80° C., and employed real-time PCR and culture to study the incidenceof Desulfovibrio in autism and controls and to recover Desulfovibriostrains to study further. We did not use three of the autistic subjects'stools because two had Asperger's syndrome and one was an adult. We alsoadded four additional healthy controls.

Subjects

The stool specimens were from 30 regressive autistic subjects, 23 malesand 7 females, with GI symptomotology (primarily constipation with orwithout “compensatory” diarrhea but abdominal distension ordiscomfort/pain were also common), 7 healthy siblings (5 females and 2males), and 12 healthy controls (7 males and 5 females, ages 4-10 years)having no contact with autistic children. Ages ranged from 2 to 13years. Eleven of the autistic subjects had severe to moderately severesymptoms and 19 bad mild disease. The autistic subjects and theirsiblings were from the practice of John Green, M.D. in Evergreen, Oreg.All subjects there had an educational or developmental pediatricianevaluation. Subsequently, Dr. Green evaluated each patient and validatedthe diagnosis of autism based on impairment in social skills, impairmentin language skills and verbal communication, sensory disturbances,repetitive stereotypical behaviors, and gastrointestinal disturbances.Dr. Green's practice has been limited to autistic spectrum disorderssince 1999 and he has evaluated and treated some 2,000 patients in thattime. The twelve control subjects were from the Los Angeles area andother areas served by CARD and were children of administrative personnelwho worked at the Center for Autism and Related Disorders (CARD) but noton floors where patients were seen. There were 7 males and 5 females andages ranged from four to ten years. The study was approved by IRBs atthe two collaborating institutions and at the VA Medical Center in LosAngeles. Subjects were excluded if they had been on any antibacterialagents or probiotics within the past month. It wasn't possible to getthe parents to agree to discontinue antifungal agents (which possiblyhave some antibacterial activity as well) and it wasn't possible tostandardize diet for all subjects.

Stool Collection, Storage, and Initial Processing

Specimens (the entire bowel movement because bacteria are distributedrandomly in feces) were collected at the homes of the children and wereshipped to the Wadsworth Va. Anaerobe Laboratory the same day, instyrofoam boxes with frozen shipping packets. Specimens always arrivedat the laboratory the next morning. They were homogenized inside ananaerobic chamber, DNA extracted, and cultures set up. Data wascorrected to dry weight.

Blinding of Subject Information

Subject identification was withheld from all investigators doinglaboratory studies. Identification of type of patient (autism vs. thetwo control groups) was withheld from laboratories involved until thedata was collected; it was then released for data analysis.

DNA Extraction

DNA was extracted using a commercial system (QIAamp DNA stool mini kit;Qiagen) according to manufacturer's instructions. Our laboratory studieshave shown that this product produces high-quality DNA free ofPCR-inhibiting substances.

Real-Time PCR

The real-time PCR procedure used is described in an earlierpublication³⁰. Oligonucleotide primers and probes were designed (seeTable 12). Standard curves were constructed to enumerate theDesulfovibrio species using the 7500 Real-Time PCR System (AppliedBiosystems).

TABLE 12 Sequences of oligonucleotide primers and probes Target Targetorganism T_(m) gene Forward primer (5′-3′) Reverse primer (5′-3′)Probe (5′-3′) D. desulfuricans 60 16S rRNA GGATCGTAAACCTCTGTCCTTTACGCCCAGT(G/A)ATTCC AACTACGTTGTGCTAATCAGCA AG GCGT D. fairfieldensis53 168 rRNA GGACTCATCCTCATACGA TCGAGTAGAGTGGCGCA GCAAGCAGAGGCCGTCTTTCCCCA CT D. intestinalis 60 16S rRNA GGATCGTAAACCTCTGTCCTTTACGCCCAGT(G/A)ATTCC AGAAACCGCACCGTGCTAATC AG AGCG D. piger 6016S rRNA GGATCGTAAACCTCTGTC CTTTACGCCCAGT(G/A)ATTCCAAGAAACTAGGGTGTTCTAATC AG ATCATCC D. vulgaris 60 16S rRNAGGATCGTAAACCTCTGTC CTTTACGCCCAGT(G/A)ATTCC CGGTGCTAATCAGCCGTGGTCT AG GDesulfovibrio 16S rRNA CCGTAGATATCTGGAGG ACATCTAGCATCCATCGTTTAC spp.AACATCAG AGC

Culture

A selective and differential medium was designed consisting of Brucellaagar with ferric ammonium citrate 0.05%, pyruvate 1%, MgSO₄ 0.25%, andvancomycin 10 μl. Plates were inoculated with 10 μl/plate of serialten-fold dilutions of each specimen, and examined at intervals for 10days. Black colonies were studied by 16S rRNA sequencing³¹ foridentification.

Antimicrobial Susceptibility Testing

The Clinical and Laboratory Standards Institute (CLSI) protocol was usedin a serial two-fold plate dilution procedure³² employing 25 strains ofDesulfovibrio isolated from stools or clinical specimens plus two typestrains, as noted, and the standard reference strains (Bacteroidesthetaiotaomicron and Bacteroides fragilis) and seven antimicrobialagents. The production of β-lactamase was detected by the nitrocefindisk test³³.

Statistical Analysis

The maximum of the four culture values for any sample (D. piger, D.fairfieldensis, D. desulfuricans, and Desulfovibrio species) was takenas the final “culture” outcome. Similarly, the maximum of the threereal-time-PCR values (D. piger, D. fairfieldensis, D. species) was takenas the final RT-PCR value. Any response not recorded as “zero” (i.e.,below the threshold of detection) is considered positive. Valuesrecorded as zero (below the level of detection) are negative.

In addition to reporting observed agreement between the culture andRT-PCR assessments for the presence or absence of Desulfovibrio species,the Kappa statistic was computed to correct for chance agreement. Kappais near zero if the agreement is only due to chance.

The p values for comparing proportions between different groups werecomputed using Fishers exact test or the chi-square test for overallcomparisons. Trends in proportions were computed using theCochran-Armitage method.

Since all of the control values, all but one or two of the sib controlvalues and a majority of the autism values are “zero” for culture andRT-PCR, mean comparisons were not carried out.

Results

Culture and real-time PCR. Stools were obtained from 30 autisticsubjects, 7 siblings of these children, and 12 healthy controls, a totalof 49 specimens studied. The results of the culture and real-time PCR,with statistical analysis, are given in Tables 13A, 13B, 13C, and 13D.Fourteen of the stool specimens from 30 autistic children were positivefor Desulfovibrio by culture or real-time PCR (46.7%) compared to twostools of seven from siblings of autistic children (28.6%) and zero of12 stools from healthy controls not exposed to autism. Severity wasarbitrarily graded from 4 (most severe) to 1 (least severe) based on Dr.Green's clinical judgment and 0 for no autism. The more severe theautism the higher the percent positive by either culture or real-timePCR; the numbers are small but the dose response was consistent.Overall, there were 13 specimens positive by culture and 9 by real-timePCR; the overall agreement between methods was significant (p=0.005).The combination of both methods increased the overall yield to 16positives in the 49 specimens studied, compared to 13 positive byculture and 9 by real-time PCR. The specificity was relatively good(90%), but the sensitivity was only 47%. The odds ratio was 7.44.

TABLE 13A Overall agreement between culture versus RT-PCR in n = 49subjects RT-PCR Neg RT-PCR Pos Total Culture Neg 33 3 36 Culture Pos 7 613 Total 40 9 49 Observed agreement = (33 + 6)/49 = 79.6%, Kappa = 0.419+/− 0.150, p = 0.005

TABLE 13B Percent positive by culture OR RT-PCR Group N Num pos Pct posSE Autism 30 14 46.7% 9.1% Control 12 0   0% — Sib control 7 2 28.6%17.1% Total 49 16 32.6% 6.7% Overall chi square = 8.55, p value = 0.014Comparison p value Autism vs control 0.003 Autism vs sib 0.675 Controlvs sib 0.123

TABLE 13C Autism severity vs percent positive by culture OR RT-PCRSeverity n Num pos Pct pos SE 0 (no autism) 19 2 10.5% 7.0% 1 6 2 33.3%19.2% 2 9 4 44.4% 16.6% 3 7 3 42.9% 18.7% 4 4 2 50.0% 25.0% >=1* 4 375.0% 21.7% Total 49 16 32.6% 6.7% *not used in trend p valuecalculation since actual severity unknown Trend Z = −2.23, p value =0.03

TABLE 13D Sensitivity and specificity using culture OR RT-PCR autismcontrol Total Test pos 14 2 16 Test neg 16 17 33 Total 30 19 49 Positiveif EITHER is positive, Negative if BOTH are negative Combining controlsand sib controls Sensitivity = 14/30 = 47% ± 9% Specificity = 17/19 =90% ± 7% Unweighted Accuracy = (Sensitivity + Specificity)/2 = 68% ± 6%.Odds ratio 7.44

Antimicrobial susceptibility. The susceptibility of Desulfovibriospecies to seven antimicrobial compounds is shown in Table 14. The tenstrains tested all produced β-lactamase.

TABLE 14 Antimicrobial susceptibility patterns of Desulfovibrio (MIC,mcg/ml) Aztreonam Azt + Clav Colistin Kanamycin Polymyxin B TMX/SulVancomycin 1 8236 256 16 >512 512 256 256 2 8381 128 16 >512 256 >512256 >512 3 8907 256 16 >512 256 >512 512 >512 4 8933 256 8 >512 512 >512256 >512 5 8951 64 16 >512 512 >512 256 >512 6 9070 512 16 >512512 >512 >512 >512 7 9120 256 32 >512 256 >512 256 >512 8 9706 25632 >512 256 >512 256 >512 9 9976 256 8 >512 128 >512 256 512 10 10831 256 4 >512 256 128 256 11 11081  256 8 >512 512 256 256 12 11094  2568 >512 512 256 >512 13 11232  64 8 >512 256 >512 128 >512 14 11346  25616 >512 512 >512 512 >512 15 11378  256 8 >512 512 256 256 16 11437  6416 >512 128 >512 4 >512 17 11568  256 8 >512 512 >512 >512 >512 1811623  256 16 >512 256 >512 512 >512 19 11717  256 8 >512 512 256 >51220 11789  32 8 >512 256 16 >512 21 11830  64 4 >512 128 128 >512 2211949  256 8 >512 128 >512 512 >512 23 12379  16 4 >512 512 8 256 2413966  24 16 >512 512 >512 512 >512 25 4019 512 8 >512 512 256 >512 26 7757^(T) 256 64 >512 512 256 >512 >512 27 29098^(T) 32 64 >512 128 256256 1

Discussion

There are 220 species of sulfate-reducing bacteria in 60 genera.¹⁴ Onlythe genus Desulfovibrio appears to be associated with autism althoughother genera such as Clostridium that contain some sulfate reducers orthat might act in other ways additively or synergistically withDesulfovibrio should be studied. Desulfovibrio is an anaerobicgram-negative non-spore-forming short rod, usually curved and rapidlymotile by means of a single polar flagellum (except for D. piger). Black(H₂S), pinpoint colonies or black confluent growth, may appear in 3 daysof anaerobic incubation on appropriate media. Colonies may continue toappear up to about 10 days of continued incubation. The five species ofDesulfovibrio found in humans are D. desulfuricans, D. fairfieldensis,D. piger, D. intestinalis, and D. vulgaris. The incidence ofDesulfovibrio in human bowel flora varies in different countries,perhaps related to diet and age¹⁵. The organisms were not only in feces;they colonized the gut wall in rectal biopsies, with counts of 10⁶-10⁷.The low incidence in our control group suggests that it is uncommon inyoung children in the U.S. The fact that the stools from siblings werein between the autism and control stools for Desulfovibrio positivity byeither culture or real-time PCR suggests the possibility of exposure ofthe siblings to the organism, although the numbers are small. However,the trend was significant (p=0.03).

Our proposed pathogenesis of regressive autism with GI manifestations isshown in FIG. 5. Genetic background and environmental contamination withtoxic substances can damage the immunologic capabilities of youngchildren, predisposing them to autism. There may be exposure toDesulfovibrio from contact with other children who are autistic, or fromthe environment (soil; foods; surfaces, fomites in the home.). Diet isimportant in determining the intestinal microbiome. The final insult maybe exposure to antimicrobial compounds (probably primarily oralcephalosporins) administered for ear or other infections. Niehus andLord (20) reviewed medical records from birth to age 2 years of 99children (75 who developed ASD, 29 who had regressive disease, and 24who developed normally). Children who developed autism had significantlymore ear infections and were given significantly more antibiotics thanthose who developed normally. Desulfovibrio is often resistant tocephalosporins which suppress certain elements of the indigenous bowelflora and thus permit outgrowth of Desulfovibrio. Also, variousbeta-lactam antibiotics inhibit the mitochondrialcarnitine/acylcarnitine transporter ¹⁶. Antibiotics of all types alsorelease LPS from bacteria¹⁷.

The onset of regressive autism in relation to use of antibiotics isreminiscent of antimicrobial use promoting Clostridium difficileinfection in predisposed elderly persons in intensive care units inhospitals, or in nursing homes. Note also the parallel with infantbotulism in which Clostridium botulinum (from soil, corn syrup, orhoney) colonizes the gastrointestinal tract of infants whose bowel florais not yet fully developed¹⁸. Antibiotics may also play a role in olderchildren and even adults in colonization of the gut with C. botulinumand subsequent clinical botulism. In our earlier studies, we wereimpressed with the possibility of involvement of clostridia, notably theC. clostridioforme “group”, in autism¹⁹ but our recent pyrosequencingstudy ¹² which included a larger number of autistic children andcontrols and permitted much more extensive detection of bowel floraelements indicated these clostridia were not prominent in autism andsuggested that Desulfovibrio might be. One other organism, Bacteroidesvulgatus, also had a significantly higher percent of the total flora inthe autistic subjects than in the controls in the pyrosequencing study¹²but real-time PCR studies indicated that the differences were really notsignificant. The pyrosequencing study also demonstrated some bacteriathat were potentially protective (higher percent of the total flora incontrol subjects than in the autistics), particularly bifidobacteria.This requires further study. Although Desulfovibrio does not producespores, it has several mechanisms making it resistant to oxygenation andable to survive other deleterious encounters²⁰. The physiology andmetabolism of Desulfovibrio position it uniquely to account for much ofthe pathophysiology seen in autistic children.

Lipopolysaccharide (LPS) has been noted in Desulfovibrio ²¹ and by Dr.Beenhouwer of our group. Emanuele et al. showed that serum levels ofendotoxin were significantly higher in autistic subjects than in healthyones and inversely and independently correlated with socialization²².

Desulfovibrio produces hydrogen sulfide which is genotoxic and, athigher concentrations, cytotoxic to the colonic epithelium. H₂S may alsocreate cellular energy deficiency by inhibiting the β-oxidation ofbutyrate²³ . Desulfovibrio competes effectively with butyrate-producingbacteria for lactate, an important electron donor for sulfate reductionby Desulfovibrio. Interestingly, some sulfate-reducing bacteria carryout a propionic acid fermentation of lactate, converting 3 molecules oflactate to 2 of propionate; MacFabe et al. found that intracerebralinjection of propionic acid or other short-chain fatty acids in rats ledto biologic, chemical, and pathologic changes characteristic ofautism¹³, Sulfide can also be derived from sulfur compounds in the dietor from endogenous mucins which are sulfated glycoproteins; this leadsto vulnerability of the colonic epithelium. H₂S is inhibitory tomitochondrial cytochrome c oxidase. The corrosive activity of hydrogensulfide on metals, an important environmental problem, may have acounterpart in human health in consequence of the importance of certainmetals in human metabolism. Hydrogen sulfide penetrates membranesreadily and is likely to penetrate colonic epithelial cells and beyond,influencing local blood flow, immune function and neural reflexactivity. Dr. Emma Allen-Vercoe offers an intriguing suggestion of atrial of 5-ASA to inhibit sulfidogenesis by Desulfovibrio.

James et al. present an excellent study of the normal methionine cycle,methylation and transsulfuration and abnormalities seen in 20 autisticchildren (19 of whom had “regressive” autism) as compared to results in33 control children²⁴. Waring and her colleagues in the United Kingdomhave also been interested in sulfur metabolism in autism²⁵. An importantpaper published recently by Yap et al. described results of urinarymetabolic phenotyping using ¹H NMR spectroscopy²⁶. Perturbation ofsulfur and amino acid metabolism was noted in autistic subjects vs.controls and there were also abnormalities in the tryptophan-nicotinicacid metabolic pathway. An increased demand for methylation of nicotinicacid to its N-methylated acid and amide, as seen in this study, wouldproduce more stress on the compromised methylation capacity of autisticchildren and would lead to increased oxidative stress.

Aztreonam, kanamycin, gentamicin and other aminoglycosides, andvancomycin are virtually unabsorbed when given by the oral route. Thesame is true for colistin and polymyxin B, but these compounds do notpenetrate the intestinal mucosa and this could be a problem foreradication of Desulfovibrio which can attach to the bowel mucosa¹⁵. Thedrugs that are not absorbed to any extent when given orally achieve veryhigh levels in the bowel; this is important in interpreting the data inTable 14. Also important to note is the fact that aztreonam is a betalactam compound and thus is susceptible to inactivation bybeta-lactamases produced by Desulfovibrio as well as by many othercomponents of the intestinal flora²⁷. This can be overcome by givingaztreonam together with a beta-lactamase inactivator such as clavulanicacid (which resulted in much lower minimal inhibitory concentrations[MICs]. The potential toxicity of colistin, polymyxin, and kanamycinwould mitigate against their use, given the availability of otheroptions. Trimethoprim/sulfamethoxazole is absorbed well on oraladministration so gut levels would likely be low which may be why thiscompound predisposes to autism, according to anecdotal reports.Vancomycin was included because of our positive experience with the oralform in an open label trial of regressive autism⁹. The drug is not veryactive on a weight basis, but it must be remembered that fecal levels ofvancomycin are often 2-5,000 mcg/gm after oral administration.

Among other data on antimicrobial susceptibility of Desulfovibrio arereports that it has been recovered from various infections, someserious²⁷⁻²⁹.

REFERENCE LIST FOR EXAMPLE 5

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Although certain presently preferred embodiments of the invention havebeen specifically described herein, it will be apparent to those skilledin the art to which the invention pertains that variations andmodifications of the various embodiments shown and described herein maybe made without departing from the spirit and scope of the invention.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.

1. A method of treating autism associated with Desulfovibrio overgrowthin the gastrointestinal tract of a patient, said method comprisingadministering to the patient suffering from said autism a treatmentcourse of aztreonam in an amount effective to treat autism in thepatient, thereby treating autism.
 2. The method claim 1, furthercomprising administering a beta-lactamase inhibitor.
 3. The method ofclaim 2, wherein the beta-lactamase inhibitor is selected from the groupconsisting of clavulanic acid, tazobactam, sulbactam and LK-157 orothers.
 4. The method of claim 1, wherein aztreonam and thebeta-lactamase inhibitor are administered concurrently.
 5. A method forscreening for compounds that inhibit Desulfovibrio and thereby inhibitautism, wherein said method comprises: a) obtaining a sample from asubject, a) contacting a sample containing Desulfovibrio with a moleculeof interest, b) determining whether contact results in inhibition ofDesulfovibrio.
 6. The method of claim 5, wherein the compound isbacteriostatic.
 7. The method of claim 5, wherein the compound isbactericidal.
 8. The method of claim 5, wherein the compound is a smallmolecule, protein, peptide, antibiotic, pre-formed antibodies,bacteriophage or a combination thereof.
 9. A screening method accordingto claim 5, which comprises separately contacting each of a plurality ofsamples to be tested.
 10. The screening method of claim 9, wherein theplurality of samples comprises more than about 10⁴ samples.
 11. Thescreening method of claim 9, wherein the plurality of samples comprisesmore than about 5×10⁴ samples.
 12. The method of claim 5, wherein thesubject is selected from the group consisting of human, monkey, ape,dog, cat, cow, horse, rabbit, mouse and rat subjects.
 13. A method ofscreening for autism comprising: a) obtaining a sample from a subject,and b) determining whether Desulfovibrio is present in the sample, thepresence of Desulfovibrio being indicative of autism.
 14. The method ofclaim 13, wherein the subject is human.
 15. The method of claim 5,wherein the sample is selected from the group consisting of feces,urine, blood and plasma.
 16. The method of claim 5, wherein inhibitionof Desulfovibrio comprises decrease in growth of Desulfovibrio,elimination of growth of Desulfovibrio or delay in growth ofDesulfovibrio.
 17. The method of claim 1 further comprisingadministering a probiotic and/or a probiotic group.
 18. The method ofclaim 17, wherein the probiotic and/or a probiotic group is selectedfrom the group consisting of bacteria that competes with Desulfovibriofor nutrients and bacteria that inhibits growth of Desulfovibrio.